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1
Kwon, Se Jeong, Dohee Ahn, Hyun-Mo Yang, Hyo Jin Kang, and Sang J. Chung. "Polyphyllin D Shows Anticancer Effect through a Selective Inhibition of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP2)." Molecules 26, no. 4 (February 5, 2021): 848. http://dx.doi.org/10.3390/molecules26040848.
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Natural products have continued to offer tremendous opportunities for drug development, as they have long been used in traditional medicinal systems. SHP2 has served as an anticancer target. To identify novel SHP2 inhibitors with potential anticancer activity, we screened a library containing 658 natural products. Polyphyllin D was found to selectively inhibit SHP2 over SHP1, whereas two other identified compounds (echinocystic acid and oleanolic acid) demonstrated dual SHP1 and SHP2 inhibition. In a cell-based assay, polyphyllin D exhibited cytotoxicity in Jurkat cells, an acute lymphoma leukemia cell line, whereas the other two compounds were ineffective. Polyphyllin D also decreased the level of phosphorylated extracellular signal-regulated kinase (p-ERK), a proliferation marker in Jurkat cells. Furthermore, knockdown of protein tyrosine phosphatase (PTP)N6 (SHP1) or PTPN11 (SHP2) decreased p-ERK levels. However, concurrent knockdown of PTPN6 and PTPN11 in Jurkat cells recovered p-ERK levels. These results demonstrated that polyphyllin D has potential anticancer activity, which can be attributed to its selective inhibition of SHP2 over SHP1.
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Zhu, Helen He, Kaihong Ji, Nazilla Alderson, Zhao He, Shuangwei Li, Linheng Li, and Gen-Sheng Feng. "Coordinated Regulation of Embryonic and Adult Hematopoietic Stem Cell Activity by PTPN11/Shp2." Blood 116, no. 21 (November 19, 2010): 2630. http://dx.doi.org/10.1182/blood.v116.21.2630.2630.
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Abstract Abstract 2630 Shp2, a tyrosine phosphatase with two SH2 domains, is implicated in malignant blood diseases. Germline dominant active mutations in PTPN11 that codes for Shp2 are found in approximately 50% of Noonan syndrome (NS) patients who have high risk of juvenile myelomonocytic leukemia (JMML). Somatic gain-of-function mutations in PTPN11 have also been detected in nonsyndromic JMML, pediatric AML, B-cell precursor ALL and myelodysplastic syndromes. The majority of leukemia-associated PTPN11 mutations disrupt the auto-inhibitory interactions between its N-terminal SH2 and the phosphatase domain, resulting in elevated catalytic activity. However, the physiological function of Shp2 in normal hematopoiesis in adults remains to be elucidated, although a Shp2 function in embryonic hematopoiesis was documented previously. Mouse embryonic stem cells (mESCs) homozygous for targeted exon 3 deletion are defective in differentiation to all blood cell lineages, suggesting its role in commitment/specification of hematopoietic stem cells (HSCs) during embryogenesis. Indeed, our previous experiments suggested a developmental blockage from mESC to mesoderm and hemangioblast differentiation by the exon 3 deletion. Notably, distinct mechanisms are involved in embryonic and adult hematopoiesis. Despite an essential role for specification of HSC fate from embryonic mesoderm, SCL/tal-1 was found dispensable for HSC maintenance and differentiation in adults. Similar observation was made for transcription factor Runx. We show here that inducible ablation of PTPN11/Shp2 in the hematopoietic compartment leads to severe cytopenia in bone marrow, spleen and periphery. Shp2 removal suppressed the number and function of HSCs and myeloid progenitors. Shp2-deficient HSCs failed to reconstitute irradiated recipient mice, due to combined defects in homing, self-renewal and survival. Therefore, PTPN11/Shp2 indeed plays an essential role in maintenance of a functional HSC pool in adult mammals. This study reveals a critical signaling component conserved in embryonic and adult hematopoiesis. Given the essential role of Shp2 in normal HSCs, developing a chemical that distinguishes wild-type and mutant Shp2 enzymes may be a therapeutic approach for elimination of leukemic stem cells (LSCs) harboring mutant PTPN11/Shp2. Disclosures: No relevant conflicts of interest to declare.
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Li, XingJun, Charles Goodwin, Zhenyun Yang, Sarah C. Nabinger, Briana Richine, Gordon Chan, Helmut Hanenberg, et al. "Macrophage NADPH Oxidase Activation and ROS Production Is Positively Regulated By Shp2 Phosphatase Function." Blood 124, no. 21 (December 6, 2014): 1397. http://dx.doi.org/10.1182/blood.v124.21.1397.1397.
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Abstract Macrophages are professional phagocytic cells, and express pattern recognition receptors such as C-type lectins and integrins for the detection of invading pathogens. Both Dectin-1 (a C-type lectin) and complement receptor 3 (CR3, a β2-integrin) are expressed on innate immune cells including macrophages, neutrophils, and dendritic cells. Dectin-1 stimulation by b-glucan-containing particles (zymosan) and CR3 stimulation by serum opsonized zymosan (SOZ) activate Erk- and Akt-dependent signaling resulting in phagocytosis and production of an oxidative burst. Shp2, a protein tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt signaling, supports hematopoietic development, and is commonly mutated in juvenile myelomonocytic leukemia (JMML). However, no studies have examined the role of Shp2 in Dectin-1- or CR3-stimulated NADPH oxidase activation or ROS production. As activation of Erk and Akt stimulates NADPH oxidase by phosphorylating p47phox, we hypothesized that Shp2 positively regulates ROS production in response to Dectin-1 or CR3 stimulation. Using murine peritoneal exudate macrophages (PEMs), both zymosan and SOZ exposure induced maximal ROS production 10 minutes post-stimulation, which corresponded to maximal induction of Shp2 phosphorylation (Y580, proposed to promote Shp2 phosphatase activity) and Erk phosphorylation. Using bone marrow derived macrophages (BMMs) from mice bearing a conditionally deleted allele of Ptpn11 (Shp2flox/flox;Mx1Cre+), ROS production was significantly reduced in response to zymosan and SOZ in Shp2flox/flox;Mx1Cre+ BMMs compared to control Shp2flox/flox;Mx1Cre- BMMs. Notably, the phagocytic index of the Shp2flox/flox;Mx1Cre+ and Shp2flox/flox;Mx1Cre- BMMs was similar, and protein components of the NADPH oxidase complex (p40phox, p67phox, and p47phox) were expressed at similar levels. To define the biochemical role of Shp2 in ROS production, we generated yellow fluorescent protein (YFP)-tagged Shp2 constructs bearing mutation of the N-SH2 (R32K) or phosphatase (C463A) domain and retrovirally expressed these constructs in murine BMMs. When subjected to zymosan or SOZ stimulation, mutation of either the N-SH2 or phosphatase domain resulted in reduced ROS production. Using time-lapse confocal videomicroscopy, we found that Shp2-R32K-YFP failed to translocate to the phagosome in SOZ-stimulated BMMs; however, phosphatase dead Shp2-C463A-YFP strongly translocated to the phagosome despite producing lower ROS levels. These findings specifically pointed to Shp2 phosphatase function as crucial in positively regulating NADPH oxidase and ROS production. Accordingly, we reasoned that macrophages expressing JMML-associated gain-of-function (GOF) Shp2 mutants, characterized to have increased phosphatase activity, would produce elevated ROS levels. As anticipated, BMMs retrovirally expressing GOF Shp2-D61Y or GOF Shp2-E76K and PEMs from mice bearing a conditionally induced gain-of-function allele of Ptpn11 (Shp2D61Y/+;Mx1Cre+) similarly produced significantly elevated levels of zymosan- and SOZ-stimulated ROS compared to WT Shp2-expressing BMMs or PEMs, respectively. Given the positive role of Shp2 phosphatase in promoting zymosan- and SOZ-stimulated ROS production, we investigated putative Shp2 substrates in response to zymosan stimulation. SHPS-1 (SH2 domain-containing protein tyrosine phosphatase substrate 1) is a myeloid inhibitory immunoreceptor expressed on macrophages, requires tyrosine phosphorylation to exert its inhibitory effect, and has been shown to be de-phosphorylated by Shp2. Consistent with its potential function in regulation ROS production, SHPS-1 is strongly associated with phagosomes in zymosan-stimulated PEMs. In immunoblot analysis, reduced phospho-SHPS-1 levels kinetically correlated with maximal zymosan-stimulated Shp2 phosphorylation and ROS production, and increased levels of phospho-SHPS-1 were found in BMMs expressing phosphatase dead Shp2-C463A compared to cells expressing WT Shp2. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated NADPH oxidase activation and ROS production in macrophages, and that mechanistically, Shp2 may exert its positive effect by de-phosphorylating and thus negatively regulating the inhibitory function of SHPS-1. Disclosures No relevant conflicts of interest to declare.
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Liu, Xia, Hong Zheng, Xiaobo Li, Siying Wang, Howard J. Meyerson, Wentian Yang, Benjamin G. Neel, and Cheng-Kui Qu. "Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies." Proceedings of the National Academy of Sciences 113, no. 4 (January 11, 2016): 984–89. http://dx.doi.org/10.1073/pnas.1508535113.
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Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.
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Chan, Gordon, Laurene S. Cheung, Wentian Yang, Michael Milyavsky, Ashley D. Sanders, Shengqing Gu, Wan Xing Hong, et al. "Essential role for Ptpn11 in survival of hematopoietic stem and progenitor cells." Blood 117, no. 16 (April 21, 2011): 4253–61. http://dx.doi.org/10.1182/blood-2010-11-319517.
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Abstract Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor protein-tyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of Ptpn11 in murine hematopoietic cells leads to bone marrow aplasia and lethality. Mutant mice show rapid loss of hematopoietic stem cells (HSCs) and immature progenitors of all hematopoietic lineages in a gene dosage-dependent and cell-autonomous manner. Ptpn11-deficient HSCs and progenitors undergo apoptosis concomitant with increased Noxa expression. Mutant HSCs/progenitors also show defective Erk and Akt activation in response to stem cell factor and diminished thrombopoietin-evoked Erk activation. Activated Kras alleviates the Ptpn11 requirement for colony formation by progenitors and cytokine/growth factor responsiveness of HSCs, indicating that Ras is functionally downstream of Shp2 in these cells. Thus, Shp2 plays a critical role in controlling the survival and maintenance of HSCs and immature progenitors in vivo.
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Romero, Celeste, Lester J. Lambert, Douglas J. Sheffler, Laurent J. S. De Backer, Dhanya Raveendra-Panickar, Maria Celeridad, Stefan Grotegut, et al. "A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors." Journal of Biological Chemistry 295, no. 9 (January 17, 2020): 2601–13. http://dx.doi.org/10.1074/jbc.ra119.010838.
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The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.
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Mohi, M. Golam, Heike Keilhack, Sarah Cohen, Christina Boulton, Toshiyuki Araki, Ifor Williams, Jeffery L. Kutok, D. Gary Gilliland, and Benjamin G. Neel. "Mechanism of Oncogenesis by Leukemia-Associated Mutants of Shp2 (PTPN11)." Blood 104, no. 11 (November 16, 2004): 199. http://dx.doi.org/10.1182/blood.v104.11.199.199.
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Abstract Juvenile Myelomonocytic Leukemia (JMML) is a clonal hematopoietic disorder of childhood that is principally characterized by proliferation of cells of the granulocytic and monocytic lineages. Previous studies showed that Ras or Nf1 mutations contribute to ~65% cases of JMML. More recently, mutations in the protein tyrosine phosphatase Shp2 (PTPN11) have been found in ~35% of sporadic JMML, and at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and childhood B-cell acute lymphoblastic leukemia (B-ALL). Interestingly, germline mutations of Shp2 cause the autosomal dominant genetic disorder Noonan Syndrome (NS) and NS patients may have an increased incidence of JMML. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and JMML mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activated. We have compared the biochemical and biological effects of NS and JMML mutations. When produced as recombinant proteins in bacteria, nearly all NS mutations studied showed increased PTP activity. However, the JMML mutations E76K and D61Y have the highest activity, resembling the fully active mutant E76A that we defined earlier. Retroviral-mediated expression of E76K or D61Y, but not wild type (WT) Shp2 in murine bone marrow (BM) cells results in cytokine-independent myeloid colony outgrowth, as well as hypersensitivity to both IL-3 and GM-CSF. Notably, NS- associated mutants (e.g., D61G or N308D) also could transform BM cells to factor-independence, but yielded far fewer colonies. Transformation by the leukemia-associated E76K mutant required the phosphatase activity and intact FLVRE motifs in the Shp2 SH2 domains. Gab2, a major Shp2 SH2 domain binding protein, also was required for transformation. When retrovirally transduced BM cells were transplanted into lethally irradiated recipients, E76K and D61Y but not WT Shp2 evoked fatal myeloproliferative disease (MPD) in 30–40% of recipients characterized by splenomegaly, leukocytosis, neutrophilia and monocytosis with occasional anemia and thrombocytopenia. Another ~20% showed less severe MPD at early stage and ultimately succumbed to T-ALL at later times. Histological analysis of mice dying MPD revealed BM and spleen packed with myeloid cells and livers with perivascular myeloid infiltrates. Flow cytometry on BM and spleen confirmed the presence of MPD. Moreover, BM from E76K and D61Y transplanted recipients exhibited enhanced factor-independent colony formation. Furthermore, mast cells derived from E76K and D61Y transplanted mice exhibit increased proliferation and enhanced activation of Erk, Akt and Stat5 in response to IL-3. In contrast, mice with a knock-in mutation of NS (D61G) displayed only a mild MPD. These data support a model in which leukemia-associated mutants of Shp2 are strong hypermorphs that enhance signaling via βc cytokines, whereas NS mutants are less severe gain of function alleles. Our results provide the first evidence that Shp2 mutants have a causal role in leukemogenesis and therefore, Shp2 is the first bona fide PTP proto-oncogene.
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Xu, Dan, Xia Liu, Wen-Mei Yu, Howard J. Meyerson, Stanton L. Gerson, and Cheng-Kui Qu. "Non-Lineage/Stage Restricted Effects of a Gain-of-Function Mutation in Tyrosine Phosphatase Ptpn11 (Shp2) on Malignant Transformation of Hematopoietic Cells." Blood 118, no. 21 (November 18, 2011): 392. http://dx.doi.org/10.1182/blood.v118.21.392.392.
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Abstract Abstract 392 SHP2, a protein tyrosine phosphatase implicated in multiple cell signaling processes, plays an essential role in hematopoietic cell development. Our previous studies have demonstrated that this phosphatase is required for erythroid, myeloid, and lymphoid development and that it functions in cytokine signaling in both catalytically-dependent and –independent manners. Notably, germline and somatic mutations (heterozygous) in PTPN11 (encoding SHP2) have been identified in 35% of the patients with juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disorder (MPD). Furthermore, PTPN11 mutations are also found in pediatric myelodysplastic syndromes (10%), B cell lymphoblastic leukemia (B-ALL) (7%), acute myeloid leukemia (AML) (4%), and sporadic solid tumors. These mutations result in hyperactivation of SHP2 catalytic activity. In addition, PTPN11 disease mutations, especially leukemia mutations, enhance the binding of mutant SHP2 to signaling partners. Although previous studies have shown that Ptpn11 mutations induce cytokine hypersensitivity in myeloid progenitors and MPD in mice, it is unclear whether Ptpn11 mutations also play a causal role in the pathogenesis of acute leukemias. If so, the underlying mechanisms and the cell origin of leukemia initiating/stem cells (LSCs) remain to be determined. PTPN11E76K mutation is the most common and most active PTPN11 mutation found in JMML and acute leukemias. However, the pathogenic effects of this mutation have not been well characterized. We created Ptpn11E76K conditional knock-in mice. Global Ptpn11E76K/+ mutation resulted in early embryonic lethality associated with enhanced ERK signaling. Induced knock-in of this mutation in pan hematopoietic cells led to MPD as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors. These animals subsequently progressed to acute leukemias. Intriguingly, in addition to AML, T-ALL and B-ALL were evolved. PTPN11E76K/+ mutation induced LSC development not only in stem cells but also in lineage committed progenitors as tissue-specific knock-in of Ptpn11E76K/+ mutation in myeloid, T lymphoid, and B lymphoid progenitors also resulted in AML, T-ALL, and B-ALL, respectively. Further analyses revealed that Shp2 was distributed to centrosomes and that Ptpn11E76K/+ mutation promoted LSC development partly by causing centrosome amplification and genomic instability. Thus, Ptpn11E76K mutation has non-lineage specific effects on malignant transformation of hematopoietic cells and initiates acute leukemias at various stages of hematopoiesis. This mutation may play an initiating role in the pathogenesis of pediatric acute leukemias. Disclosures: No relevant conflicts of interest to declare.
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Liu, Wei, Xia Liu, and Cheng-Kui Qu. "Critical Role of the Gab2/PI3K/mTOR Pathway in the Pathogenesis of Ptpn11 (Shp2) Mutation-Induced Myeloproliferative Disease." Blood 120, no. 21 (November 16, 2012): 2856. http://dx.doi.org/10.1182/blood.v120.21.2856.2856.
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Abstract Abstract 2856 Germline and somatic mutations (heterozygous) in Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, have been identified in juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disease (MPD), and pediatric acute leukemias. These mutations cause hyperactivation of Shp2 catalytic activity and enhance the binding of mutant Shp2 to signaling partners. Ptpn11 mutations are sufficient to drive the development of JMML-like MPD and acute leukemias in mice, suggesting that they play a causal role in the pathogenesis of hematological malignancies. However, the mechanisms by which Ptpn11 mutations induce these malignancies are not completely understood and the signaling partners that mediate the pathogenic effects of Ptpn11 mutations have not been explored. We previously generated a line of conditional knock-in mice with Ptpn11E76K mutation, the most common and most active Ptpn11 mutation found in JMML and acute leukemias. Induced knock-in of this mutation in hematopoietic cells resulted in MPD with full penetrance as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors (J. Exp. Med., 2011). Recently, we discovered that the interaction between Shp2 E76K and Gab2, a prominent interacting protein of Shp2 and a scaffolding protein important for cytokine-induced PI3K/Akt/mTOR signaling, was greatly enhanced, and that mTOR was highly activated in Ptpn11E76K/+ MPD cells. To address the role of Gab2 and mTOR in the pathogenesis of Ptpn11E76K/+ mutation-induced MPD, Ptpn11E76K/+/Gab2-/- double mutant mice were generated and their phenotypes were compared with those of Ptpn11E76K/+ single mutant mice. MPD phenotypes were markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice. Overproduction of myeloid cells in the bone marrow was alleviated, and splenomegaly was diminished in the double mutants. Myeloid cell infiltration in the liver also decreased. Cytokine (IL-3 and GM-CSF) sensitivity of myeloid progenitors was significantly decreased in Ptpn11E76K/+/Gab2−/− mice as compared to that in Ptpn11E76K/+ mice. Hyperactivation of HSCs and excessive myeloid differentiation caused by Ptpn11E76K mutation were largely corrected by deletion of Gab2. Furthermore, we treated Ptpn11E76K/+ mice with Rapmycin, a specific and potent mTOR inhibitor, which substantially diminished MPD phenotypes. Collectively, this study reveals the essential role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic effects of Ptpn11E76K/+ mutation and suggests that Gab2 and mTOR are potential therapeutic targets for the treatment of Ptpn11-associated hematological malignancies. Disclosures: No relevant conflicts of interest to declare.
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Mohi, M. Golam, and Benjamin G. Neel. "The role of Shp2 (PTPN11) in cancer." Current Opinion in Genetics & Development 17, no. 1 (February 2007): 23–30. http://dx.doi.org/10.1016/j.gde.2006.12.011.
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Chan, Gordon, Demetrios Kalaitzidis, and Benjamin G. Neel. "The tyrosine phosphatase Shp2 (PTPN11) in cancer." Cancer and Metastasis Reviews 27, no. 2 (February 20, 2008): 179–92. http://dx.doi.org/10.1007/s10555-008-9126-y.
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Traina, Fabiola, João Agostinho Machado Neto, Arthur Antolini, Flavio Araújo Borges, Mariana Lazarini, Patricia M. B. Favaro, Leticia Fröhlich -Archangelo, et al. "IRS1 and SHP2 Signaling in Myelodysplastic Syndrome and Acute Myeloid Leukemia." Blood 114, no. 22 (November 20, 2009): 1779. http://dx.doi.org/10.1182/blood.v114.22.1779.1779.
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Abstract Abstract 1779 Poster Board I-805 Introduction Myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The occurrence of acute leukemia results from a combination of mutations and changes in protein functions that confer the ability of proliferation, defects in cell differentiation and apoptosis. The deregulation of multiple cellular signaling pathways in MDS and acute leukemia hinders the development of an effective drug for treatment. SHP2 is a cytoplasmic tyrosine phosphatase encoded by the gene PTPN11. The SHP2 signaling pathway is involved in important functions as regulation of proliferation, differentiation and cell migration and has been described as overexpressed and constitutively phosphorylated in acute and chronic leukemic cells. The IGF1/IRS1 signaling pathway has an important role in the development of various cancers such as breast, colon and prostate cancer and multiple myeloma. Increased expression of IRS1 has been described in neoplastic transformation, and reduction of IRS1 expression has been related to the level of differentiation of neoplastic cells and with a poor prognosis in solid tumors. In acute lymphoblastic leukemia (ALL), IRS1 expression was noted to be overexpressed and correlated with prognosis. The expression of SHP2 and IRS1 in MDS has not yet been elucidated, as well the expression of IRS1 in AML. The aim of the present study was to investigate the gene and protein expression of IRS1 and SHP2 in MDS and AML patients, and also PTPN11 exon 3 mutations. Patients and Methods We studied 06 healthy donors, 26 patients with MDS (18 low-risk [RA/RARS] and 07 high-risk [RAEB/RAEBt] according to FAB classification), 24 with AML and 5 with ALL. Samples were obtained from bone marrow. Gene expression was evaluated by real time RT-PCR in total cells. Western blot of mononuclear cells was performed for protein expression. PTPN11 mutations were analyzed by PCR with specific primers and sequencing. Results Real time RT-PCR demonstrated a reduced expression of IRS1 in MDS cells (0.13 [0.01-1.19], P=0.01) and AML cells (0.10 [0.009-7.14], P=0.02), when compared to normal cells (0.62 [0.10-1.41]). On the other hand, IRS1 expression was significantly increased in LLA samples (2.35[0.73-11.64] P=0.03) when compared to normal cells. Western blot analysis confirmed the gene expression pattern: IRS1 expression and phosphorylation were down-regulated in MDS and AML and upregulated in ALL when compared to normal hematopoietic cells. Regarding PTPN11 expression, no difference was detected in MDS (1.03 [0.07-4.96], P >0.05) and AML cells (0.48 [0.17-2.29], P >0.05) when compared with normal hematopoietic cells (0.56 [0.2-1.0]). Interesting, PTPN11 expression was increased in low risk MDS (1.35 [0.12-4.96], P=0.048) or total MDS (1.03 [0.07-4.96], P =0.039) when compared to AML cells. Western blot analysis confirmed the SHP2 expression pattern. However, SHP2 phosphorylation was marked increased in both MDS and AML compared to normal hematopoietic cells. PTPN11 mutation was not detected in the samples. Conclusions Our results show that IRS1 expression and phosphorylation is down-regulated in MDS and AML patients compared to normal hematopoietic cells, suggesting that IGF1/IRS1 signaling pathway is differently regulated in normal cells, MDS, AML and ALL. IRS1 overexpression in ALL is in agreement with previous authors and indicates IRS1 as a possible target in ALL, thought not MDS and AML. SHP2 activation is increased in MDS and AML, and was independent from PTPN11 mutation. The increased activation of SHP2 in MDS, not previously described, indicates that this tyrosine phosphatase may play a role in MDS pathophysiology and leukemia transformation. Keywords: IRS1, SHP2, leukemia, myelodysplasia Disclosures No relevant conflicts of interest to declare.
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Chen, Lili, Wei Chen, Maria Mysliwski, Justin Serio, James Ropa, Fardokht Abulwerdi, Rebecca J. Chan, et al. "Hyperactive Ptpn11 Mutations Alters Leukemic Stem Cell Frequency through Mcl-1 Overexpression." Blood 124, no. 21 (December 6, 2014): 3565. http://dx.doi.org/10.1182/blood.v124.21.3565.3565.
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Abstract Shp2 is a non-receptor protein-tyrosine phosphatase encoded by PTPN11 and implicated in the Ras, JAK-STAT and PI3K pathways. Activating mutations in Shp2 are found in patients with developmental disorders such as Noonan and LEOPARD syndrome, as well as, hematologic malignancies. Although rare in most other solid tumors, Shp2 mutations are common in juvenile myelomonocytic leukemia (JMML) accounting for ~35% of cases. To understand its role as a cooperating mutation in AML we sequenced PTPN11 in human samples. Here we report that Shp2 mutations are present in human AML at a rate of 6.6% (6/91) in the ECOG E1900 dataset. To investigate the biological function of Shp2 mutations we asked how this functions in a cooperative model of leukemogenesis with the MLL-AF9 fusion protein. Despite showing increased genetic stability compared to other leukemias, MLL leukemias commonly contain type I mutations that can functionally cooperate resulting in more aggressive leukemias. These mutations often occur in genes encoding components of the Ras pathway including mutually exclusive mutations of NRAS, KRAS, PTPN11 and NF1 and account for ~37% of MLL rearranged leukemias. However, the mechanisms of cooperation with MLL fusion proteins are poorly understood. We found that the Shp2E76K activating mutation commonly found in humans significantly accelerates MLL-AF9 mediated leukemogenesis. The E76K mutation results in structural changes that confer increased phosphatase activity to the Shp2 protein and increased Ras signaling. We attribute the MLL-AF9/Shp2E76K cooperation to a more rapid leukemic initiation as evidenced in colony formation assays using mouse bone marrow HSPCs. Cells transduced with MLL-AF9/Shp2E76K expanded faster than MLL-AF9 cells at early stages following transduction, indicating more efficient transformation of myeloid progenitors than MLL-AF9 alone. Cytokine independent growth is achieved in MLL-AF9 cells following expression of Shp2E76K through the constitutive activation of the IL3 signaling pathway and ERK phosphorylation. Importantly, addition of Shp2E76K significantly accelerated MLL-AF9 mediated acute myeloid leukemia in mice, indicating activated Shp2 cooperates with MLL-AF9 in vivo. In addition, leukemic stem cell frequency was increased by greater than 4 fold due to Shp2E76K expression. As Shp2 is reported to regulate an anti-apoptotic gene program, we investigated these in the context of MLL-AF9 leukemic cells with and without Shp2E76K. While Bcl2, BclXL and Mcl-1, were upregulated in Shp2E76K cells, Mcl-1 showed the highest upregulation in response to Shp2E76K. Further, expression of Mcl-1 with MLL-AF9 in colony assays phenocopies expression of Shp2E76K suggesting that, mechanistically, Shp2 mutations may cooperate through activation of an anti-apoptotic gene program, primarily through Mcl-1. Finally, we asked how leukemic cells bearing Shp2E76K would respond to small molecule inhibition of Mcl-1. MLL-AF9 leukemic cells expressing Shp2E76K were desensitized to small molecule mediated Mcl-1 inhibition consistent with increased Mcl-1 protein. These data were confirmed in human cells where U937 cells, which contain an activating Shp2 mutation, exhibited resistance to Mcl-1 inhibition compared to ML2 or K562 cell which both bear wild type Shp2. Together, these data suggest patients with hyperactive Shp2 signaling may respond poorly to drugs targeting Mcl-1 due to an overabundance of the protein. Disclosures No relevant conflicts of interest to declare.
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Schramm, Christine, Deborah M. Fine, Michelle A. Edwards, Ashley N. Reeb, and Maike Krenz. "The PTPN11 loss-of-function mutation Q510E-Shp2 causes hypertrophic cardiomyopathy by dysregulating mTOR signaling." American Journal of Physiology-Heart and Circulatory Physiology 302, no. 1 (January 2012): H231—H243. http://dx.doi.org/10.1152/ajpheart.00665.2011.
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The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.
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Xu, Rongzhen, Yingzi Yu, Shu Zheng, Xiaoying Zhao, Qinghua Dong, Zhiwen He, Yun Liang, et al. "Overexpression of Shp2 tyrosine phosphatase is implicated in leukemogenesis in adult human leukemia." Blood 106, no. 9 (November 1, 2005): 3142–49. http://dx.doi.org/10.1182/blood-2004-10-4057.
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Abstract Shp2 tyrosine phosphatase plays a critical role in hematopoiesis, and dominant active mutations have been detected in the human gene PTPN11, encoding Shp2, in child leukemia patients. We report here that although no such mutations were detected in 44 adult leukemia patients screened, Shp2 expression levels were significantly elevated in primary leukemia cells and leukemia cell lines, as compared with normal hematopoietic progenitor cells. The Shp2 protein amounts correlated well with the hyperproliferative capacity but were inversely associated with the differentiation degree of leukemia cells. Suppression of Shp2 expression induced apoptosis and inhibition of leukemic cell clonogenic growth. Notably, the majority of Shp2 was preferentially localized to the plasma membrane and was constitutively phosphorylated on tyrosine in leukemia cells, and also in normal hematopoietic cells following mitogenic stimulation. Based on these results, we propose that aberrantly increased expression of Shp2 may contribute, collaboratively with other factors, to leukemogenesis.
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Li, Shuangwei, Diane DiFang Hsu, Hongyang Wang, and Gen-Sheng Feng. "Dual faces of SH2-containing protein-tyrosine phosphatase Shp2/PTPN11 in tumorigenesis." Frontiers of Medicine 6, no. 3 (August 6, 2012): 275–79. http://dx.doi.org/10.1007/s11684-012-0216-4.
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Idrees, Muhammad, Lianguang Xu, Seok-Hwan Song, Myeong-Don Joo, Kyeong-Lim Lee, Tahir Muhammad, Marwa El Sheikh, Tabinda Sidrat, and Il-Keun Kong. "PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development." Cells 8, no. 10 (October 18, 2019): 1272. http://dx.doi.org/10.3390/cells8101272.
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This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.
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Li, Liang, Hardik Modi, Tinisha McDonald, John Rossi, Jiing-Kuan Yee, and Ravi Bhatia. "A critical role for SHP2 in STAT5 activation and growth factor–mediated proliferation, survival, and differentiation of human CD34+ cells." Blood 118, no. 6 (August 11, 2011): 1504–15. http://dx.doi.org/10.1182/blood-2010-06-288910.
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Abstract SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34+ cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34+ progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.
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Yang, Zhenyun, Takako Kondo, Cara S. Voorhorst, Sarah C. Nabinger, Leila Ndong, Fuqin Yin, Edward M. Chan, et al. "Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants." Molecular and Cellular Biology 29, no. 16 (June 15, 2009): 4376–93. http://dx.doi.org/10.1128/mcb.01330-08.
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ABSTRACT Juvenile myelomonocytic leukemia (JMML) is characterized by myelomonocytic cell overproduction and commonly bears activating mutations in PTPN11. Murine hematopoietic progenitors expressing activating Shp2 undergo myelomonocytic differentiation, despite being subjected to conditions that normally support only mast cells. Evaluation of hematopoietic-specific transcription factor expression indicates reduced GATA2 and elevated c-Jun in mutant Shp2-expressing progenitors. We hypothesized that mutant Shp2-induced Ras hyperactivation promotes c-Jun phosphorylation and constitutive c-Jun expression, permitting, as a coactivator of PU.1, excessive monocytic differentiation and reduced GATA2. Hematopoietic progenitors expressing activating Shp2 demonstrate enhanced macrophage CFU (CFU-M) compared to that of wild-type Shp2-expressing cells. Treatment with the JNK inhibitor SP600125 or cotransduction with GATA2 normalizes activating Shp2-generated CFU-M. However, cotransduction of ΔGATA2 (lacking the C-terminal zinc finger, needed to bind PU.1) fails to normalize CFU-M. NIH 3T3 cells expressing Shp2E76K produce higher levels of luciferase expression directed by the macrophage colony-stimulating factor receptor (MCSFR) promoter, which utilizes c-Jun as a coactivator of PU.1. Coimmunoprecipitation demonstrates increased c-Jun-PU.1 complexes in mutant Shp2-expressing hematopoietic progenitors, while chromatin immunoprecipitation demonstrates increased c-Jun binding to the c-Jun promoter and an increased c-Jun-PU.1 complex at the Mcsfr promoter. Furthermore, JMML progenitors express higher levels of c-JUN than healthy controls, substantiating the disease relevance of these mechanistic findings.
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Zhu, Helen He, Xiaolin Luo, Kaiqing Zhang, Jian Cui, Huifang Zhao, Zhongzhong Ji, Zhicheng Zhou, et al. "Shp2 and Pten have antagonistic roles in myeloproliferation but cooperate to promote erythropoiesis in mammals." Proceedings of the National Academy of Sciences 112, no. 43 (October 12, 2015): 13342–47. http://dx.doi.org/10.1073/pnas.1507599112.
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Previous data suggested a negative role of phosphatase and tensin homolog (Pten) and a positive function of SH2-containing tyrosine phosphatase (Shp2)/Ptpn11 in myelopoiesis and leukemogenesis. Herein we demonstrate that ablating Shp2 indeed suppressed the myeloproliferative effect of Pten loss, indicating directly opposing functions between pathways regulated by these two enzymes. Surprisingly, the Shp2 and Pten double-knockout mice suffered lethal anemia, a phenotype that reveals previously unappreciated cooperative roles of Pten and Shp2 in erythropoiesis. The lethal anemia was caused collectively by skewed progenitor differentiation and shortened erythrocyte lifespan. Consistently, treatment of Pten-deficient mice with a specific Shp2 inhibitor suppressed myeloproliferative neoplasm while causing anemia. These results identify concerted actions of Pten and Shp2 in promoting erythropoiesis, while acting antagonistically in myeloproliferative neoplasm development. This study illustrates cell type-specific signal cross-talk in blood cell lineages, and will guide better design of pharmaceuticals for leukemia and other types of cancer in the era of precision medicine.
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Zhu, Helen He, Kaihong Ji, Nazilla Alderson, Zhao He, Shuangwei Li, Wen Liu, Dong-Er Zhang, Linheng Li, and Gen-Sheng Feng. "Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool." Blood 117, no. 20 (May 19, 2011): 5350–61. http://dx.doi.org/10.1182/blood-2011-01-333476.
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Abstract The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment, and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However, it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2, acting downstream of Kit and other RTKs, promotes Kit gene expression, constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM, spleen, and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors, and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing, self-renewal, and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore, this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals, at least in part through a kinase-phosphatase-kinase cascade.
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Bellio, Marie, Cédric Garcia, Thomas Edouard, Sophie Voisin, Benjamin G. Neel, Cendrine Cabou, Philippe Valet, et al. "Catalytic dysregulation of SHP2 leading to Noonan syndromes affects platelet signaling and functions." Blood 134, no. 25 (December 19, 2019): 2304–17. http://dx.doi.org/10.1182/blood.2019001543.
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Abstract Src homology 2 domain–containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbβ3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress–dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding.
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Goodwin, Charles B., Zhenyun Yang, Sasidhar Vemula, Fuqin Yin, Reuben Kapur, and Rebecca J. Chan. "Genetic Disruption of the PI3K Regulatory Subunit, p85α Partially Normalizes Gain-of-Function PTPN11-Induced Hypersensitivity to GM-CSF in Hematopoietic Progenitors." Blood 114, no. 22 (November 20, 2009): 3968. http://dx.doi.org/10.1182/blood.v114.22.3968.3968.
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Abstract Abstract 3968 Poster Board III-904 Juvenile Myelomonocytic Leukemia (JMML) is a lethal myeloproliferative disorder (MPD) of children, characterized by hyperproliferation of myelomonocytic cells and hypersensitivity to Granulocyte-Monocyte Colony-Stimulating Factor (GM-CSF). Most patients show hyperactivation of Ras via one or more mutations, including in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. It has been demonstrated that gain-of-function mutant Shp2 (Shp2 E76K and Shp2 D61Y) causes hyperactivation of the Mitogen-Activated Protein Kinase (MAPK) pathway. Additionally, we have previously shown that macrophage progenitor cells transduced with Shp2 D61Y or Shp2 E76K showed elevated levels of phospho-Akt, both at baseline and following 1 hour of GM-CSF stimulation, indicating a role for the Phospho-Inositol-3-Kinase (PI3K)/Akt pathway in the phenotype of elevated proliferation and survival in mutant Shp2-expressing cells (Yang, et al, 2008). However, it remains to be elucidated how PI3K contributes to the phenotype of increased proliferation and survival in cells bearing gain-of-function mutations in Shp2. Class IA PI3K is a lipid kinase heterodimer consisting of one of three catalytic subunits (p110α, p110β, or p110δ) and one of two regulatory subunits (p85α or p85β). It has been demonstrated that knocking out the main regulatory subunit, p85α, abrogated the hyperproliferative phenotype in mast cell progenitors bearing an oncogenic mutation in Kit in a model of another MPD, Systemic Mastocytosis (Munugalavadla, et al, 2007). In order to examine whether eliminating expression of p85α would cause a similar correction in cells expressing gain-of-function mutant Shp2, we performed timed matings of mice heterozygous for the knock-out of Pik3r1, which encodes the p85α subunit as well as its isoforms, p55α and p50α, since homozygous Pik3r1-/- is lethal in utero. WT and Pik3r1-/- fetal liver cells were isolated from 14.5 day embryos and transduced with either WT Shp2 or mutant Shp2 E76K. Transduced cells were subjected to serum deprivation followed by a 24-hour treatment with increasing doses of GM-CSF, and proliferation was then measured with H3-thymidine incorporation assays. We found that the absence of all the Pik3r1 isoforms resulted in a significant but incomplete correction of GM-CSF hypersensitivity in Shp2 E76K-expressing cells. To further investigate this observation, WT Pik3r1 and Pik3r1-/- macrophage progenitors, transduced with WT Shp2 or mutant Shp2 E76K as described above, were serum- and growth factor-deprived and then stimulated for 1 hour with GM-CSF. Western blot analysis showed that there was an expected increase in phospho-Akt in WT Pik3r1 cells following GM-CSF stimulation and that this increase was larger in Shp2 E76K-expressing cells than in WT Shp2-expressing cells, as previously observed. Upon genetic disruption of Pik3r1, Akt activation was reduced in both WT Shp2- and Shp2 E76K-expressing cells; however, the phospho-Akt in the Shp2 E76K-expressing cells was not reduced to WT levels. The phospho-Akt levels mirrored the proliferation pattern displayed by these cells in the H3-thymidine incorporation assays, where a modest reduction in proliferation in Pik3r1-/-, Shp2 E76K cells corresponded to the modest reduction in phospho-Akt levels in the same cells. Additionally, we found that Pik3r1-/-, Shp2 E76K cells also showed a blunted increase in phospho-Erk levels following GM-CSF stimulation compared to the WT Pik3r1, Shp2 E76K cells, indicating that knocking out Pik3r1 affects the MAPK pathway as well, which likely also contributes to the reduction in proliferation seen in Pik3r1-/-, Shp2 E76K cells following GM-CSF stimulation. Based on these data, we conclude that: (1) gain-of-function Shp2 activity results in dysregulated PI3K signaling, contributing to the leukemic phenotype of increased proliferation and survival; (2) PI3K signaling is reduced but not completely normalized in the absence of the main regulatory subunit, p85α and its isoforms, in gain-of-function mutant Shp2-expressing cells; and (3) there is cross-talk between the PI3K and MAPK pathways in the presence of Shp2 activating mutations, which is revealed by knocking out Pik3r1. Disclosures: No relevant conflicts of interest to declare.
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Yang, Zhenyun, Takako Kondo, Cara s. Voorhorst, Sarah C. Nabinger, Leila Ndong, Fuqin Yin, Edward m. Chan, et al. "Increased C-Jun and Reduced GATA2 Expression Promotes Aberrant Monocytic Differentiation and Expansion Induced by Activating PTPN11 Mutants." Blood 112, no. 11 (November 16, 2008): 3720. http://dx.doi.org/10.1182/blood.v112.11.3720.3720.
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Abstract Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation and expansion despite being subjected to conditions that normally support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression using quantitative RT-PCR indicates equal expression of PU.1 in mutant Shp2- and WT Shp2-expressing hematopoietic progenitors; however, GATA2 expression is substantially reduced and c-Jun expression is increased in mutant Shp2-expressing progenitors. Based on these findings, we hypothesized that mutant Shp2-induced Ras hyperactivation produces constitutive c-Jun expression permitting, as a co-activator of PU.1, excessive monocytic differentiation and reduced GATA2 expression. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (amino acids 330 – 407, DGATA2), which is needed to compete with c-Jun for binding to PU.1, would normalize mutant Shp2-induced monocytic differentiation and expansion. To examine this hypothesis, we utilized retroviral co-transduction of bone marrow Lin-enriched cells to generate six experimental groups: pMIEG3-Shp-2WT plus pCD4 (empty vector); pMIEG3-Shp2WT plus pCD4-GATA2; pMIEG3-Shp2WT plus pCD4-ΔGATA2; pMIEG3-Shp-2E76K plus pCD4; pMIEG3-Shp-2E76K plus pCD4-GATA2; pMIEG3-Shp-2E76K plus pCD4-ΔGATA2. Cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2 mutant E76K and a secondary empty vector produced significantly more CFU-M than cells expressing WT Shp2. Upon co-transduction with GATA2, the number of CFU-M generated from E76K-expressing cells was significantly reduced. In contrast, cotransduction of ΔGATA2 lacking the C-terminal zinc finger failed to normalize the number of CFU-M produced by E76K-expressing cells. Mechanistically, we reasoned that if the c-Jun-PU.1 interaction contributes to the mutant Shp2-induced aberrant monocytic differentiation, we would observe increased expression directed by the macrophage colony-stimulating factor receptor (MCSFR) promoter, originally characterized to be activated by PU.1 and co-activator c-Jun. Consistently, MCSFR promoter-directed luciferase expression exhibited higher levels in NIH3T3 cells expressing Shp2E76K compared to those expressing WT Shp2. Furthermore, co-immunoprecipitation assays using nuclear extracts demonstrate increased c-Jun-PU.1 complexes in mutant Shp2-expressing hematopoietic progenitors, while chromatin immunoprecipitation assays demonstrate an increased association of the c-Jun-PU.1 complex at the MCSFR promoter. Moreover, CD34+ JMML progenitors express significantly higher levels of c-JUN than CD34+ cord blood progenitors from healthy newborns, substantiating the disease relevance of these mechanistic findings. These data suggest that one fundamental sequela of Ras hyperactivation in mutant Shp2-expressing cells is elevated c-Jun expression and an increased c-Jun-PU.1 interaction promoting monocytic differentiation and, thus, overproduction of myelomonocytic cells, the hallmark of JMML. These findings imply that pharmaceutical interventions that inhibit c-Jun expression or function could theoretically inhibit mutant Shp2-induced monocytic differentiation and, thus, serve as a novel approach for treatment of JMML.
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Ruess, Dietrich, Guus Heynen, Katrin Ciecielski, Walter Birchmeier, Roland Schmid, and Hana Algül. "Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase." Pancreatology 18, no. 4 (June 2018): S91. http://dx.doi.org/10.1016/j.pan.2018.05.246.
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Ruess, Dietrich A., Guus J. Heynen, Katrin J. Ciecielski, Jiaoyu Ai, Alexandra Berninger, Derya Kabacaoglu, Kivanc Görgülü, et al. "Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase." Nature Medicine 24, no. 7 (May 28, 2018): 954–60. http://dx.doi.org/10.1038/s41591-018-0024-8.
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Liu, Wei, Xia Liu, Wen-Mei Yu, Kevin D. Bunting, and Cheng-Kui Qu. "Critical Role of Stat5 in the Maintenance of Leukemic Stem Cells in Ptpn11-Associated Juvenile Myelomonocytic Leukemia." Blood 124, no. 21 (December 6, 2014): 818. http://dx.doi.org/10.1182/blood.v124.21.818.818.
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Abstract Effective therapeutic interventions for juvenile myelomonocytic leukemia (JMML), a fatal childhood malignancy, are lacking. Relapse is the most frequent cause of treatment failure, most likely due to the persistence of leukemic stem cells (LSCs), a small population of self-renewing precursor cells that give rise to the bulk of tumor cells. This reservoir of tumor cells are responsible for long-term maintenance of leukemia growth, and are also a major source of drug resistance. Clearly, a novel approach focused on the unique properties of LSCs is needed. However, it remains a critical challenge how such cells may be eradicated. JMML is known to be caused by genetic mutations in cell signaling proteins involved in the Ras pathway, among which the phosphatase Ptpn11 (Shp2) is most frequently mutated. Ptpn11 mutations (heterozygous) cause greatly increased catalytic activity. We have recently created conditional knock-in mice with the Ptpn11E76K mutation, the most common Ptpn11 mutation found in JMML. Induction of the Ptpn11E76K/+ mutation in these mice (Ptpn11E76K/+/Mx1-Cre+) leads to JMML-like myeloid malignancy with full penetrance by aberrant activation of stem cells and myeloid progenitors. In an effort to understand the biological properties of LSCs in JMML, we studied leukemic hematopoietic stem cells (Lin-Sca-1+c-Kit+Flk2-CD150+CD48- cells)(referred to as LSCs since they reproduce the same disease in sub-lethally-irradiated transplants) in this mouse model. We found that Stat5 was hyper-activated in LSCs in Ptpn11E76K/+/Mx1-Cre+ mice in the chronic phase. Stat5 hyperactivation is likely to be mediated through Jak2 kinase as Jak2 is highly activated in Shp2 E76K-expressing cells. More importantly, these LSCs appear to rely on hyperactivation of Stat5 for maintenance and self-renewal because deletion of Stat5ab in Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl double mutant mice resulted in massive cell death in LSCs while neither Stat5ab knockout nor Ptpn11E76K/+ knock-in alone mice had these stem cell phenotypes. Apoptotic cells in the stem cell population were 6.38±1.42, 4.06±0.51, 10.52±6.88, and 24.50±10.27% in Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, Stat5abfl/fl/Mx1-Cre+, and Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice, respectively. Consequently, the stem cell pool in Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl double mutants was drastically decreased. Numbers of stem cells per femur were 3.36±1.51, 0.94±0.63, 1.15±0.39, and 0.17±0.20 (x103) in Ptpn11+/+/Mx1-Cre+, Ptpn11E76K/+/Mx1-Cre+, Stat5abfl/fl/Mx1-Cre+, and Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice, respectively. Ptpn11E76K/+/Mx1-Cre+/Stat5abfl/fl mice died of pan cytopenia within 4-8 weeks of Stat5 deletion while none of other groups of mice did. Repopulation capabilities of the double mutant stem cells were essentially lost in sub-lethally-irradiated recipient animals. Further mechanistic investigations revealed that tyrosine phosphorylation levels (indicative of activity) of Stat3, one of the substrates of the Shp2 phosphatase, were decreased by ~7.50 fold in Ptpn11E76K/+ LSCs as compared to Ptpn11+/+ control cells due to the enhanced dephosphorylation by the hyperactive Shp2 E76K mutant. Thus, diminished Stat3 activity sensitized Ptpn11E76K/+ LSCs to Stat5 depletion-induced cell death. Taken together, this study suggests a crucial role of Stat5 in the maintenance of LSCs in Ptpn11-associated JMML. The synthetic lethality induced by loss of Stat5 in Ptpn11-mutated JMML raises the possibility that clinically-used inhibitors of upstream Jak2 kinase may be effective in eradicating LSCs in this particular subtype of JMML. Disclosures No relevant conflicts of interest to declare.
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Chen, Xiaolan, Yuhui Huang, Ling Yan, Marie Brault, Zhizhang Wang, Maryann T. Arildsen, and Elizabeth Yang. "p19Arf Cooperates with Shp2 in Lymphoid Leukemogenesis." Blood 112, no. 11 (November 16, 2008): 3809. http://dx.doi.org/10.1182/blood.v112.11.3809.3809.
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Abstract Shp2 gain-of-function mutations are found in human acute lymphoblastic leukemia, as well as in juvenile myelomonocytic leukemia and acute myeloid leukemias. Genetic data from Noonan Syndrome patients bearing mutations in PTPN11/Shp2 suggest that Shp2 mutation alone is insufficient for leukemogenesis, and other genetic alterations are required. We investigated E76K Shp2-induced leukemogenesis in p19Arf+/− and p19Arf−/− backgrounds. Wild type mice reconstituted with Arf+/− or Arf−/− bone marrow cells (BMCs) transduced with E76K Shp2 developed hematologic disease with increased incidence and severity than mice reconstituted with vector-transduced Arf+/− or Arf−/− BMCs, with E76K Shp2-transduced Arf−/− BMCs causing the most aggressive disease at the highest rate. E76K Shp2 expression in Arf+/− BMCs caused myeloproliferation consisting of mainly Mac1+Gr1+ cells associated with extramedullary hematopoiesis, but E76K Shp2 expression in Arf−/− BMCs caused a shift in disease phenotype to transplantable B220+ leukemias. Consistently, E76K Shp2 transduced Arf+/− or Arf−/− BMCs from reconstituted pre-leukemic mice showed increased proB cell populations compared to vector transduced BMCs, suggesting that the E76K Shp2 expressing proB cells are the leukemia precursors. While Shp2 is well known to confer GM-CSF hypersensitivity in methylcellulose cultures, we found that E76K Shp2 transduced BMCs were also hypersensitive to the lymphoid colony-stimulating factor IL-7, and E76K Shp2 expressing lymphoid BMCs formed colonies in the absence of exogenous growth factor. These data suggest that gain-of-function Shp2 confers transformation features to B cell precursors and that Arf deletion potentiates E76K Shp2 in lymphoid leukemogenesis.
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Kan, Chen, Fan Yang, and Siying Wang. "SHP2-Mediated Signal Networks in Stem Cell Homeostasis and Dysfunction." Stem Cells International 2018 (June 10, 2018): 1–10. http://dx.doi.org/10.1155/2018/8351374.
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Stem cells, including embryonic stem cells (ESCs) and adult stem cells, play a central role in mammal organism development and homeostasis. They have two unique properties: the capacity for self-renewal and the ability to differentiate into many specialized cell types. Src homology region 2- (SH2-) containing protein tyrosine phosphatase 2 (SHP-2), a nonreceptor protein tyrosine phosphatase encoded by protein tyrosine phosphatase nonreceptor type 11 gene (PTPN11), regulates multicellular differentiation, proliferation, and survival through numerous conserved signal pathways. Gain-of-function (GOF) or loss-of-function (LOF) SHP2 in various cells, especially for stem cells, disrupt organism self-balance and lead to a plethora of diseases, such as cancer, maldevelopment, and excessive hyperblastosis. However, the exact mechanisms of SHP2 dysfunction in stem cells remain unclear. In this review, we intended to raise the attention and clarify the framework of SHP2-mediated signal pathways in various stem cells. Establishment of integrated signal architecture, from ESCs to adult stem cells, will help us to understand the changes of dynamic, multilayered pathways in response to SHP2 dysfunction. Overall, better understanding the functions of SHP2 in stem cells provides a new avenue to treat SHP2-associated diseases.
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Rehman, Ashfaq Ur, Mueed Ur Rahman, Muhammad T. Khan, Shah Saud, Hao Liu, Dong Song, Pinky Sultana, Abdul Wadood, and Hai-Feng Chen. "The Landscape of Protein Tyrosine Phosphatase (Shp2) and Cancer." Current Pharmaceutical Design 24, no. 32 (January 15, 2019): 3767–77. http://dx.doi.org/10.2174/1381612824666181106100837.
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Role of Shp2: The dysregulation of cell signaling cascades associated with the cell differentiation and growth, due to the deletion, insertion or point mutation in specific amino acids which alters the intrinsic conformation of the protein, can ultimately lead to a fatal cancer disease. The protein tyrosine phosphatase has been recognized as a key regulator of extracellular stimuli such as cytokine receptor and receptor tyrosine kinase signaling. In the last era, the PTPN11 gene (encode a Shp2 protein) and its association with acute myeloid, juvenile myelomonocytic, and B-cell acute lymphoblastic leukemia, Noonan syndrome, and myelodysplastic have been recognized as the cause of such deadly disease due to the occurrence of germline mutations in the interface of PTP and SH2 domain. Conclusion: The current study was designed to focus on the allosteric regulation (autoinhibition) of the of Shp2 protein. Subsequently, it will cover the last 10-year recap of Shp2 protein, their role in cancer, and regulation in numerous ways (allosteric regulation).
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Maher, GeoffreyJ, and Anne Goriely. "Teasing apart the multiple roles of Shp2 (Ptpn11) in spermatogenesis." Asian Journal of Andrology 22, no. 1 (2020): 122. http://dx.doi.org/10.4103/aja.aja_79_19.
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Zhang, Q., A. Shibani, B. Sadikovic, C. J. Howlett, and L. C. Ang. "An aggressive multifocal primary CNS histiocytosis with PTPN11 (Shp2) mutation." Neuropathology and Applied Neurobiology 44, no. 2 (February 2018): 240–43. http://dx.doi.org/10.1111/nan.12404.
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Bard-Chapeau, Emilie A., Shuangwei Li, Jin Ding, Sharon S. Zhang, Helen H. Zhu, Frederic Princen, Diane D. Fang, et al. "Ptpn11/Shp2 Acts as a Tumor Suppressor in Hepatocellular Carcinogenesis." Cancer Cell 19, no. 5 (May 2011): 629–39. http://dx.doi.org/10.1016/j.ccr.2011.03.023.
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Chan, Gordon, Demetrios Kalaitzidis, M. Golam Mohi, Wentian Yang, Jeffery Kutok, and Benjamin Neel. "Inducible Expression of Leukemia-Associated Shp2 (Ptpn11) Affects Multiple Stages of Hematopoiesis and Causes a Fatal Myeloproliferative Disorder (MPD) in Mice." Blood 110, no. 11 (November 16, 2007): 1529. http://dx.doi.org/10.1182/blood.v110.11.1529.1529.
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Abstract Germ-line mutations in ptpn11, which encodes the protein tyrosine phosphatase Shp2, cause ∼50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ∼35% of sporadic JMML; nearly all other cases have either activating Ras mutations or homozygous Nf1 deficiency. Shp2 mutations are also found at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and B-cell acute lymphoblastic leukemia (B-ALL). NS and leukemia-associated Shp2 mutations can affect the same residue, but result in different substitutions. We showed previously that the leukemia-associated mutants E76K or D61Y, but not wild type (WT), Shp2 transform bone marrow (BM) or fetal liver cells. Transplantation of E76K- or D61Y-transduced BM evoked invasive MPD in ∼60% of recipients, with most dying at 6–7 months. In this model, mutant Shp2 is expressed under retroviral promoter control and the phenotype is variable and incompletely penetrant. To assess the effect of expressing a leukemogenic Shp2 allele under endogenous promoter control, we generated knock-in mice that can conditionally express Shp2D61Y (LSL-Shp2D61Y). Global expression of the D61Y heterozygous allele was embryonic lethal. Post-natal expression of D61Y, induced by treating Mx-1 Cre; LSL-Shp2D61Y mice with pI-pC, evoked fatal MPD with ∼50% mice dying within 5–7 months. Mutant mice showed a marked increase in WBC, expansion of the Gr-1+/Mac-1+population in BM and spleen, and histopathological evidence of infiltrating MPD. Unlike in our retroviral transduction/transplant model (and similar to JMML patients), most induced LSL-Shp2D61Y mice were also anemic. Mutant mice exhibited a marked depletion of quiescent LSK (Lin−Sca1+cKit+) cells in the BM, with a concomitant increase in LSK cells in the spleen. Interestingly, Shp2 mutant-expressing BM failed to promote long term reconstitution in BM transplant assays. Cells from mutant spleens did have some long term multi-lineage reconstitution activity, but the level was substantially less than predicted by their LSK content. Moreover, no transplant recipients develop MPD. In contrast to these effects on the stem cell compartment, D61Y directly allowed cytokine-independent differentiation of CMP and GMP in vitro. Macrophages from mutant mice showed enhanced GM-CSF-mediated proliferation and ERK activation. Our results showed that expression of leukemia-associated Shp2 at endogenous levels is sufficient to evoke MPD and has cell type-specific effect on different stages of hematopoiesis.
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Yang, Zhenyun, Cara S. Voorhorst, Leila Ndong, Fuqin Yin, Takako Kondo, Eri Hashino, and Rebecca J. Chan. "The C-Terminal Zinc Finger of GATA2 Is Necessary for Normalization of Aberrant Myelomonocytic Differentiation Induced by Activating PTPN11 Mutations." Blood 110, no. 11 (November 16, 2007): 3177. http://dx.doi.org/10.1182/blood.v110.11.3177.3177.
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Abstract Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation despite being subjected to conditions that typically support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression revealed that GATA2 expression, needed for mast cell differentiation, is dramatically reduced, while, surprisingly, PU.1 expression is unchanged in cells expressing activating Shp2 mutants. In addition to lineage-specific transcription factors such as PU.1, however, c-jun also promotes monocytic differentiation by functioning as a co-activator of PU.1. Thus, we hypothesized that activating Shp2 mutations (Shp2D61Y or Shp2 E76K) induce increased c-jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression in hematopoietic progenitors. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (GATA2del330–407), which is needed for disruption of the PU.1-c-jun interaction, would normalize aberrant myelomonocytic differentiation induced by activating Shp2 mutants. Consistent with our hypothesis, quantitative RT-PCR studies revealed 5-fold higher c-jun levels in cells expressing Shp2D61Y or Shp2E76K compared to cells expressing WT Shp2. We next utilized retroviral co-transduction of murine bone marrow LDMNCs to generate six groups: pMIEG3-Shp2WT plus pCD4 (empty vector); pMIEG3-Shp2WT plus pCD4-GATA2; pMIEG3-Shp2WT plus pCD4-GATA2del330-407; pMIEG3-Shp2E76K plus pCD4; pMIEG3-Shp2E76K plus pCD4-GATA2; and pMIEG3-Shp-2E76K plus pCD4-GATA2del330-407. Transduced cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2E76K plus pCD4 produced significantly more CFU-M than cells co-transduced with WT Shp2 plus pCD4. Upon co-transduction with GATA2, the number of CFU-M generated from Shp2E76K-expressing cells was significantly reduced and was similar to that observed in cells expressing WT Shp2. In contrast, co-transduction of GATA2del330-407 failed to normalize the number of CFU-M produced by Shp2E76K-expressing cells. Quantitative RT-PCR verified ectopic GATA2 and GATA2del330-407 expression in the co-transduced cells. These findings demonstrate that restoration of GATA2 expression normalizes the propensity toward monocytic differentiation induced by Shp2E76K. The lack of correction conferred by GATA2del330-407 in combination with the observed increased c-jun expression support a model in which GATA2 and c-jun compete for binding to PU.1 to direct cell differentiation decisions in hematopoietic progenitors bearing activating Shp2 mutants. Collectively, these findings imply that normalization of transcription factor expression may provide a novel approach to differentiation-mediated therapy in JMML.
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Goodwin, Charles B., Raghuveer Mali, Gordon Chan, Benjamin Neel, Brian Lannutti, Reuben Kapur, and Rebecca J. Chan. "Phosphatidylinositol-3-Kinase p110d Uniquely Promotes Gain-Of-Function PTPN11-Induced GM-CSF Hypersensitivity In a Model Of Juvenile Myelomonocytic Leukemia." Blood 122, no. 21 (November 15, 2013): 3678. http://dx.doi.org/10.1182/blood.v122.21.3678.3678.
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Abstract Juvenile myelomonocytic leukemia (JMML) is a fatal leukemia affecting children under the age of 4 years and is characterized by myelomonocytic cell overproduction and hypersensitivity to GM-CSF. The only curative therapy is allogeneic stem cell transplantation; however, half of children relapse after this aggressive therapy. Approximately 85% of JMML patients bear loss-of-function (LOF) mutations in NF1 or CBL or gain-of-function (GOF) mutations in KRAS, NRAS, or PTPN11. Typically, these mutations are non-overlapping, with the net effect being Ras hyperactivation. Children bearing somatic GOF mutations within PTPN11, which encodes the protein tyrosine phosphatase, Shp2, exhibit the poorest prognosis. GOF Shp2 (Shp2D61Y and Shp2E76K) induces hyperactivation of both the Ras-MEK- Erk and PI3K-Akt pathways. While the Ras-MEK-Erk pathway clearly contributes to the pathogenesis of JMML, we hypothesize that the PI3K-Akt pathway cooperates with the Ras-MEK-Erk pathway to promote JMML. Recently published work indicates that genetic disruption of the PI3K regulatory subunit, p85a, reduces GOF Shp2-induced hypersensitivity to GM-CSF. However, as PI3K regulatory subunits cannot be easily inhibited pharmacologically, we examined the contribution of class IA PI3K catalytic subunits in GOF Shp2-induced JMML. Shp2 D61Y/+ ;Mx1Cre+ mice were crossed with mice bearing conditional knockout of p110a (Pik3caflox/flox) or bearing a kinase dead mutant of p110d (Pik3cdD910A/D910A). Shp2D61Y/+;Mx1Cre-, Shp2D61Y/+;Mx1Cre+, Shp2D61Y/+;Mx1Cre+; Pik3caflox/flox, and Shp2D61Y/+;Mx1Cre+; Pik3cdD910A/D910A mice were treated with polyI;polyC, and 8 weeks post-treatment, animals were euthanized followed by evaluation of spleen size, hypersensitivity of bone marrow low density mononuclear cells (LDMNCs) to GM-CSF, frequency of bone marrow phenotypically-defined common myeloid, granulocyte-monocyte, and megakaryocyte-erythroid progenitors (CMPs, GMPs, and MEPs), and GM-CSF-stimulated Erk and Akt activation. Genetic disruption of p110a failed to normalize GOF Shp2-induced splenomegaly, GM-CSF hypersensitivity in proliferation assays and methylcellulose-based progenitor assays, or hyperphosphorylation of Erk or Akt. In contrast, genetic ablation of p110d kinase activity significantly reduced spleen size, normalized progenitor hypersensitivity to GM-CSF, and reduced both Akt and Erk hyperactivation. Additionally, genetic inhibition of p110d normalized the skewed hematopoietic progenitor distribution reported in the Shp2D61Y/+;Mx1Cre+ mice, while genetic disruption of p110a failed to do so. This unique function of p110d in the context of GOF Shp2-expressing mice is significant, as p110d expression is restricted to hematopoietic cells and p110d bears transforming properties independent of Ras. While previously published work indicates that the PI3K p110a and p110d inhibitor, GDC-0941, inhibits proliferation of GOF Shp2-expressing cells, we tested if the potent p110d-specific inhibitor, GS-9820, is similarly effective. GOF Shp2-expressing bone marrow LDMNCs treated with GS-9820 demonstrated significantly reduced proliferation in a dose-dependent fashion, while GS-9820 failed to inhibit the proliferation of WT Shp2-expressing cells. GS-9820 treatment decreased Akt phosphorylation (S473 and T308) as well as reduced Erk phosphorylation, indicating that p110d inhibition also reduces signaling within the Ras-MEK-Erk pathway. While PI3K activates the canonical Akt-mTORC1 pathway, it also positively feeds back to the Ras-MEK-Erk pathway via activation of Rac-Pak-MEK; therefore, we evaluated if p110d inhibition adds to or is redundant with MEK inhibition. Treatment of GOF Shp2-expressing hematopoietic cells with the MEK inhibitor, PD0325901, effectively reduced proliferation, and addition of GS-9820 further significantly reduced proliferation, indicating that p110d works cooperatively with MEK to promote GOF Shp2-induced disease. Collectively, our findings suggest that PI3K catalytic subunit p110d functions in a Ras-MEK-Erk pathway-independent manner to promote GOF Shp2-induced hypersensitivity to GM-CSF, and suggest that PI3K p110d inhibition in combination with MEK inhibition may be a novel, optimal approach for the treatment of JMML. Disclosures: No relevant conflicts of interest to declare.
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Ishida, Hidekazu, Shigetoyo Kogaki, Jun Narita, Hiroaki Ichimori, Nobutoshi Nawa, Yoko Okada, Kunihiko Takahashi, and Keiichi Ozono. "LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 4 (October 2011): H1531—H1539. http://dx.doi.org/10.1152/ajpheart.00216.2011.
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LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.
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Jiang, X., H. Guo, J. Wu, Q. He, Y. Li, M. Wang, H. Pan, et al. "Critical role of SHP2 (PTPN11) signaling in germinal center-derived lymphoma." Haematologica 99, no. 12 (September 5, 2014): 1834–45. http://dx.doi.org/10.3324/haematol.2014.106401.
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Chan, Rebecca J., and Gen-Sheng Feng. "PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase." Blood 109, no. 3 (October 19, 2006): 862–67. http://dx.doi.org/10.1182/blood-2006-07-028829.
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Abstract Elucidation of the molecular mechanisms underlying carcinogenesis has benefited tremendously from the identification and characterization of oncogenes and tumor suppressor genes. One new advance in this field is the identification of PTPN11 as the first proto-oncogene that encodes a cytoplasmic tyrosine phosphatase with 2 Src-homology 2 (SH2) domains (Shp2). This tyrosine phosphatase was previously shown to play an essential role in normal hematopoiesis. More recently, somatic missense PTPN11 gain-of-function mutations have been detected in leukemias and rarely in solid tumors, and have been found to induce aberrant hyperactivation of the Ras-Erk pathway. This progress represents another milestone in the leukemia/cancer research field and provides a fresh view on the molecular mechanisms underlying cell transformation.
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Pandey, Garima, Nathan Horvat, Narmin E. Amin, Afua A. Akuffo, Christelle Colin, Pearlie K. Epling-Burnette, and Gary W. Reuther. "RMC-4550, an Allosteric Inhibitor of SHP2, Displays Therapeutic Efficacy in Pre-Clinical Models of Myeloproliferative Neoplasms." Blood 134, Supplement_1 (November 13, 2019): 4198. http://dx.doi.org/10.1182/blood-2019-128937.
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Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are JAK2-driven disorders resulting from mutations in JAK2, MPL, or CALR. Ruxolitinib, the only FDA-approved JAK2 inhibitor for MPNs, alleviates patient symptomology and improves quality of life, but has little effect on reducing mutant allele burden. This persistent survival of MPN cells in the face of ruxolitinib, as well as other JAK2 inhibitors that have been clinically tested, is a major clinical bottleneck to the development of an effective targeted therapy for MPN patients. Identifying new therapeutic targets which play critical roles in MPN cells and/or in JAK2 inhibitor persistence may lead to improved MPN therapies. SHP2 is an oncogenic tyrosine phosphatase that is an effector of growth factor and cytokine receptor signaling. SHP2 plays a critical role in the activation of the RAS-ERK pathway and regulates JAK-STAT signaling via numerous phosphatase-dependent mechanisms. Activating mutations of SHP2(PTPN11) have been identified in leukemia, including 8% of MPN patients whose disease progressed to acute myeloid leukemia (AML). In addition, SHP2 has been shown to mediate adaptive resistance to targeted therapies in several cancers. Given the role of SHP2 in cytokine and JAK-STAT signaling, we envisaged a potential role of SHP2 in MPN cell growth and/or survival and ruxolitinib persistence. Treatment of JAK2-V617F-driven MPN model cell lines (UKE1, SET2, and BaF3-JAK2-V617F) with ruxolitinib blocked constitutive tyrosine phosphorylation of SHP2, including phosphorylation of Y542, a marker for activated SHP2. This phosphorylation, however, was restored in ruxolitinib persistent cells. Combination treatment of the allosteric SHP2 inhibitor RMC-4550 (Revolution Medicines) with ruxolitinib prevented the development of ruxolitinib persistent cells and pre-established persistent cells remained sensitive to SHP2 inhibition. RMC-4550 treatment led to significantly reduced levels of pERK consistent with the role of SHP2 in RAS signaling. Interestingly, pERK levels in persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells suggesting pERK was more dependent on SHP2 in ruxolitinib persistent cells. SHP2 inhibitor treatment increased pSTAT5(Y694) in drug naïve cells but this increase was not observed in similarly treated persistent cells. Furthermore, while ruxolitinib inhibited pERK levels in UKE1 and SET2 cells, pERK levels recovered within 24 hrs of treatment. SHP2 inhibition prevented the recovery of pERK in the presence of ruxolitinib. Collectively, these data suggest that signaling pathways in MPN cells treated with ruxolitinib can become rewired, gaining greater dependence on SHP2, concomitant with sustained pERK and cell survival/growth. Interestingly, we identified a known activating SHP2 mutation (F71L) in UKE1 cells obtained from two independent sources - consistent with the presence of PTPN11 mutations in post-MPN AML. The persistent survival of UKE1 cells in ruxolitinib was antagonized by CRISPR-mediated reduction of SHP2 expression, providing further evidence that SHP2 contributes to ruxolitinib persistence. To assess the effects of a SHP2 inhibitor on MPN progression in vivo, we employed the MPLW515Lbone marrow transplant mouse model of MPN. Initial assessment of therapeutic treatment of mice with an established MPN phenotype indicated that once daily treatment of RMC-4550 (10 or 30 mg/kg) antagonized the MPN phenotype. Complete blood counts indicated a significant reduction in white blood cells, monocytes, and neutrophils compared to vehicle treated mice, while flow cytometry analysis indicated RMC-4550 diminished CD11b+ cell numbers to near that observed in mice transplanted with MPLWT-transduced bone marrow. RMC-4550 improved the overall health of diseased mice, as indicated by increased weight, and significantly reduced organomegaly of the spleen and liver compared to vehicle treated MPN mice. Finally, erythropoietin independent erythroid colony formation of JAK2V617F-positive MPN patient cells was suppressed following SHP2 inhibition, which synergized or enhanced the inhibition induced by ruxolitinib in this assay. In summary, our results suggest that SHP2 inhibition may represent a potential MPN therapy in both ruxolitinib naïve and resistant patients and is an attractive therapeutic target for future clinical investigation. Disclosures Epling-Burnette: Incyte Corporation: Research Funding; Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding. Reuther:Incyte Corporation: Research Funding.
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Cai, Zhigang, Chi Zhang, Jonathan J. Kotzin, Adam Williams, Jorge Henao-Mejia, and Reuben Kapur. "Role of lncRNA Morrbid in PTPN11(Shp2)E76K-driven juvenile myelomonocytic leukemia." Blood Advances 4, no. 14 (July 22, 2020): 3246–51. http://dx.doi.org/10.1182/bloodadvances.2020002123.
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Abstract Mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, contribute to ∼35% of cases of juvenile myelomonocytic leukemia (JMML). A common clinical picture in children with JMML is that it presents as a constitutive hyperinflammatory syndrome, partially reminiscent of chronic myelomonocytic leukemia in adults. Thus, a component of JMML is associated with a hyperinflammatory state and abundant innate immune cells such as neutrophils and monocytes. Recently, we showed that the evolutionarily conserved mouse lncRNA Morrbid is specifically expressed in myeloid cells and uniquely represses the expression of the proapoptotic gene Bim to regulate the lifespan of myeloid cells. However, its role in JMML has not been investigated. In this study, we characterized the role of Morrbid and its target Bim, which are significantly dysregulated in Shp2E76K/+-bearing myeloid cells, in driving JMML. Loss of Morrbid in a mouse model of JMML driven by the Shp2E76K/+ mutation resulted in a significant correction of myeloid and erythroid cell abnormalities associated with JMML, including overall survival. Consistently, patients with JMML who had PTPN11, KRAS, and NRAS mutations and high expression of MORRBID manifested poor overall survival. Our results suggest that Morrbid contributes to JMML pathogenesis.
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Ryu, Hyun-Hee, TaeHyun Kim, Jung-Woong Kim, Minkyung Kang, Pojeong Park, Yong Gyu Kim, Hyopil Kim, et al. "Excitatory neuron–specific SHP2-ERK signaling network regulates synaptic plasticity and memory." Science Signaling 12, no. 571 (March 5, 2019): eaau5755. http://dx.doi.org/10.1126/scisignal.aau5755.
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Mutations in RAS signaling pathway components cause diverse neurodevelopmental disorders, collectively called RASopathies. Previous studies have suggested that dysregulation in RAS–extracellular signal–regulated kinase (ERK) activation is restricted to distinct cell types in different RASopathies. Some cases of Noonan syndrome (NS) are associated with gain-of-function mutations in the phosphatase SHP2 (encoded by PTPN11); however, SHP2 is abundant in multiple cell types, so it is unclear which cell type(s) contribute to NS phenotypes. Here, we found that expressing the NS-associated mutant SHP2D61G in excitatory, but not inhibitory, hippocampal neurons increased ERK signaling and impaired both long-term potentiation (LTP) and spatial memory in mice, although endogenous SHP2 was expressed in both neuronal types. Transcriptomic analyses revealed that the genes encoding SHP2-interacting proteins that are critical for ERK activation, such as GAB1 and GRB2, were enriched in excitatory neurons. Accordingly, expressing a dominant-negative mutant of GAB1, which reduced its interaction with SHP2D61G, selectively in excitatory neurons, reversed SHP2D61G-mediated deficits. Moreover, ectopic expression of GAB1 and GRB2 together with SHP2D61G in inhibitory neurons resulted in ERK activation. These results demonstrate that RAS-ERK signaling networks are notably different between excitatory and inhibitory neurons, accounting for the cell type–specific pathophysiology of NS and perhaps other RASopathies.
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Kontaridis, Maria I., Kenneth D. Swanson, Frank S. David, David Barford, and Benjamin G. Neel. "PTPN11(Shp2) Mutations in LEOPARD Syndrome Have Dominant Negative, Not Activating, Effects." Journal of Biological Chemistry 281, no. 10 (December 23, 2005): 6785–92. http://dx.doi.org/10.1074/jbc.m513068200.
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Yu, Bing, Wei Liu, Wen-Mei Yu, Mignon L. Loh, Shawn Alter, Olgun Guvench, Alexander D. MacKerell, Li-Da Tang, and Cheng-Kui Qu. "Targeting Protein Tyrosine Phosphatase SHP2 for the Treatment of PTPN11-Associated Malignancies." Molecular Cancer Therapeutics 12, no. 9 (July 3, 2013): 1738–48. http://dx.doi.org/10.1158/1535-7163.mct-13-0049-t.
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Patel, Roshni, Baskar Ramdas, Lisa Deng, Victoria Jideonwo-Auman, Rebecca J. Chan, and Reuben Kapur. "Combined Pharmacological Inhibition of Bruton's Tyrosine Kinase (BTK) and Phosphoinositide 3-Kinase (PI3K) p110δ Rescues Monocytosis, Thrombocytopenia, and Splenomegaly in a Genetic Mouse Model for JMML." Blood 132, Supplement 1 (November 29, 2018): 2623. http://dx.doi.org/10.1182/blood-2018-99-118802.
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Abstract Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN). It has no effective treatments and the only treatment that prolongs survival is allogeneic hematopoietic stem cell transplant (HSCT). However, even with this aggressive intervention, approximately 50% of patients relapse with JMML within five years. The most commonly mutated gene in JMML patients is PTPN11, which encodes for the non-receptor protein tyrosine phosphatase SHP2. Gain-of-function (GOF) mutations in SHP2 lead to hyperactive RAS signaling and PTPN11 is recognized as a well-established oncogene in various leukemia's. Our lab has shown that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase (PI3K), plays an important role downstream of SHP2-signaling. Recently, we have shown that in vivo treatment with a specific pharmacologic p110δ inhibitor significantly prolongs the overall survival and reduces the splenomegaly seen in GOF Shp2-expressing mice. Having observed the effectiveness of PI3K p110δ inhibition in correcting the mutant Shp2-induced leukemia phenotype in vitro and in prolonging survival of mice in vivo, we next explored signaling molecules with which p110δ may be interacting to promote the aberrant Shp2 signaling in myeloid cells. In recent years, a key player in B cell receptor (BCR) signaling, Bruton's tyrosine kinase (BTK), has come under intense study in the field of lymphocytic leukemia and lymphoma research. Ibrutinib, a small molecule inhibitor targeting BTK, has proven to be very effective and has received FDA-approval for the treatment of a variety of B cell malignancies. Although research on BTK up to this point has focused on B cell malignancies, BTK is also highly expressed in myeloid cells and mice lacking BTK are defective in many myeloid cell functions. Thus, it is possible that BTK signaling may also play an important role in myeloid malignancies, such as JMML. Given the collaboration of BTK and p110δ in BCR signaling, the key role of p110δ in GOF Shp2-induced leukemia, and the high expression of BTK in myeloid cells, we hypothesized that BTK and p110δ function cooperatively in GOF Shp2-expressing myeloid cells to promote MPN. To test this hypothesis, we examined the potential collaboration of GM-CSF-stimulated BTK and p110δ in GOF Shp2-expressing myeloid cells and tested the inhibitor combination in vivo. We show that BTK cooperates with p110δ to promote GOF Shp2-induced leukemia. We show a role for B cell adaptor for PI3K (BCAP) in BTK upregulation of PI3K activity. In mutant Shp2 macrophages, BCAP phosphorylation is increased specifically in the larger isoforms regulating PI3K activation, and BTK inhibition results in a dose-dependent reduction in this phosphorylation. Our findings show that the MPN caused by GOF Shp2 is due to cooperative signaling between p110δ and BTK, which forms a positive feedback loop with BCAP, thus leading to more Akt/Erk hyperphosphorylation and hyperproliferation in response to GM-CSF. Given these in vitro observations, we treated GOF Shp2 bearing mice with a combination of a PI3K p110δ-specific inhibitor and, a BTK-specific inhibitor, or single agents alone and performed hematopoietic analysis. The combination treatment scheme completely rescued monocytosis and uniquely ameliorated thrombocytopenia compared to single agent treatment of diseased mice. In addition, splenomegaly was also completely rescued in all three treatment groups compared to vehicle. Detailed flow cytometric analysis of bone marrow hematopoietic stem and progenitor cell populations revealed significant repression of LSK, HPC1 and granulocyte macrophage progenitors (GMPs) with a concomitant increase in the megakaryocyte precursors (MKPs) in drug combination treated mice vs. other groups. Consistent with these observations, a significant reduction in mature myeloid cells was noted in the spleen and a complete rescue in the production of peripheral blood platelets was observed in the mutant mice treated with a combination of the two drugs. Thus, combination therapy with PI3K p110δ- and BTK-specific inhibitors profoundly rescues disease state hallmarks of JMML, and may warrant further clinical investigation. Disclosures No relevant conflicts of interest to declare.
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Mohi, M. Golam, and Benjamin G. Neel. "Structural and Signaling Requirements for Transformation Mediated by Shp2 Mutants." Blood 106, no. 11 (November 16, 2005): 3508. http://dx.doi.org/10.1182/blood.v106.11.3508.3508.
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Abstract The SH2-containing tyrosine phosphatase Shp2 (PTPN11) is required for normal growth factor and cytokine signaling. Germline Shp2 mutations cause ~50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ~35% of sporadic JMML, and at lower incidence in various chronic and acute leukemias. JMML patients without Shp2 mutations have either activating Ras mutations or homozygotic inactivation of Nf1. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and leukemia mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activating. We previously showed that leukemia-associated mutants E76K or D61Y, but not wild type (WT) Shp2 could transform bone marrow (BM) or fetal liver cells and give rise to cytokine-independent myeloid colony formation. Leukemia-associated mutants yielded much higher number and larger size of colonies than NS-associated mutants (e.g., N308D, N308S, E76D) whereas, mutants associated with both NS and leukemia (T73I, E139D, Q506P) evoked intermediate numbers. Transplantation of E76K− or D61Y− (but not parental virus or WT Shp2) transduced BM into lethally irradiated recipients evoked a fatal JMML-like disorder. To understand the structural requirements of Shp2 for transformation, we made a series of second site mutations in Shp2 E76K and expressed these mutants in murine BM. Mutation of the canonical arginine residue to methionine in the FLVRES motif in either N-SH2 or C-SH2 domain of Shp2 E76K ablated myeloid transforming ability. Mutation of the conserved arginine residue within the Shp2 E76K phosphatase domain signature motif to methionine, which diminishes phosphatase activity, also eliminated myeloid transformation. Tyrosine residues 542 and 580 in the C-terminus of Shp2 are known to undergo phosphorylation in response to many growth factors and required for Grb2 binding. Mutation of tyrosine 542 to phenylalanine markedly reduced transformation, whereas tyrosine 580 mutation alone had little effect. However, mutation of both tyrosines eliminated Shp2 E76K-evoked transformation, suggesting that Grb2-binding to Shp2 could be important for transformation. We observed that Gab2, a major Shp2 SH2 domain binding protein, was absolutely required for transformation by Shp2 E76K but not activating RasV12. Gab3, another member of Gab family protein mainly expressed in hematopoietic cells, was not critical for Shp2 E76K-evoked transformation. We also found that Stat5 was important for Shp2 E76K-mediated transformation. Mast cells derived from E76K− and D61Y− transplanted mice BM exhibited increased proliferation and enhanced activation of Erk, p38MAPK, Akt and Stat5 in response to IL-3. These results provide some mechanistic insights into how Shp2 mutants could transform primary myeloid cells and result in JMML-like MPD.
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Edouard, Thomas, Jean-Philippe Combier, Audrey Nédélec, Sophie Bel-Vialar, Mélanie Métrich, Francoise Conte-Auriol, Stanislas Lyonnet, et al. "Functional Effects of PTPN11 (SHP2) Mutations Causing LEOPARD Syndrome on Epidermal Growth Factor-Induced Phosphoinositide 3-Kinase/AKT/Glycogen Synthase Kinase 3β Signaling." Molecular and Cellular Biology 30, no. 10 (March 22, 2010): 2498–507. http://dx.doi.org/10.1128/mcb.00646-09.
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ABSTRACT LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3β (GSK-3β) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3β hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3β stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.
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Alhumaid, Muhned S., Majed J. Dasouki, Syed O. Ahmed, Halah AbalKhail, Samya Hagos, Salma Wakil, and Shahrukh K. Hashmi. "Comprehensive Genomic Analysis of Noonan Syndrome and Acute Myeloid Leukemia in Adults: A Review and Future Directions." Acta Haematologica 143, no. 6 (2020): 583–93. http://dx.doi.org/10.1159/000505715.
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Acute myeloid leukemia (AML) in the setting of Noonan syndrome (NS) has been reported before without clear guidelines for treatment or prognosis in these subgroups of patients, most likely due to its rarity and incomplete understanding of the pathogenesis of both diseases. In the current era of next-generation sequencing-based genomic analysis, we can better identify patients with NS with more accurate AML-related prognostic markers. Germline mutations in <i>PTPN11</i> are the most common cause of NS. Somatic mutations in <i>NPM1</i> occur frequently in AML. Here, we describe a young adult patient with a novel combined germline <i>PTPN11</i> and somatic <i>NPM1</i>, <i>IDH1</i>,<i></i>and<i> BCL6</i> mutations who presented with fatal AML. In addition, a 50.5-Mb interstitial deletion of 7q21.11-q33 in tumor DNA was detected by chromosomal microarray analysis. While mutations in the transcriptional repressor <i>BCL6</i> are known to contribute to the pathogenesis of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), its novel identification in this patient suggests an expanded role in aggressive AML. The identification of key molecular aberrations including the overexpression of SHP2, which drives leukemogenesis and tumorigenesis, has led to the development of novel investigational targeted SHP2 inhibitors.
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Liu, Wei, Bing Yu, Gang Xu, Wei-Ren Xu, Mignon L. Loh, Li-Da Tang, and Cheng-Kui Qu. "Identification of Cryptotanshinone as an Inhibitor of Oncogenic Protein Tyrosine Phosphatase SHP2 (PTPN11)." Journal of Medicinal Chemistry 56, no. 18 (September 4, 2013): 7212–21. http://dx.doi.org/10.1021/jm400474r.
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Han, Tao, Dai-Min Xiang, Wen Sun, Na Liu, Huan-Lin Sun, Wen Wen, Wei-Feng Shen, et al. "PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients." Journal of Hepatology 63, no. 3 (September 2015): 651–60. http://dx.doi.org/10.1016/j.jhep.2015.03.036.
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