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

A common Shp2 mutation leads to myeloproliferative disease and malignant acute leukemia in stem cells and committed progenitors, associated with Shp2 maintaining chromosomal stability

Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment

Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway, are found in 50% of patients with Noonan syndrome. These patients have an increased risk of developing leukaemia, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1β and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.

SHP-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by EGCG.

Green tea polyphenol, epigallocatechin-3-gallate (EGCG) differentially regulates the cellular growth of cancer cells in a p53-dependent manner through apoptosis and/or cell cycle arrest. In an effort to further elucidate the mechanism of differential growth regulation by EGCG, we have investigated the role of the tyrosine phosphatase, SHP-2. Comparing the responses of mouse embryonic fibroblasts (MEFs), expressing either WT or functionally inactive/truncated SHP-2, we find that inactivation of SHP-2 remarkably sensitizes cells to EGCG-mediated killing. MEFs lacking functional SHP-2 undergo massive apoptosis upon treatment with EGCG. By comparing gene expression profiles, we have identified a set of transcriptional targets of p53 that are differentially modulated in cells undergoing apoptosis. Western blot and real-time PCR analyses of a select group of genes further confirm that the expression is SHP-2-dependent. Similar observations were made in MEFs lacking p53, confirming that the expression of these “p53 target genes” is p53-independent. In addition, EGCG treatment induced the expression of p73 mRNA and protein in both cell types, but not p63. Inactivation of p73 in cells expressing nonfunctional SHP-2 markedly inhibited apoptosis and p53 target gene expression. Although phosphorylation of JNK is differentially regulated by SHP2, it was found to be dispensable for EGCG-induced apoptosis and p53 target gene expression. Our results have identified SHP-2 as a negative regulator of EGCG-induced-apoptosis and have identified a subset of p53 target genes whose expression is paradoxically not mediated by p53 but by one of its family members, p73.

Effects of a Leukemia-associated Gain-of-Function Mutation of SHP-2 Phosphatase on Interleukin-3 Signaling

Mutations in SHP-2 phosphatase that cause hyperactivation of its catalytic activity have been identified in human leukemias, particularly juvenile myelomonocytic leukemia, which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners and that overexpression (5–6-fold) of wild type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, we investigated effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction by comparing with those of overexpressed WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increased interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and phosphatidylinositol 3-kinase (PI3K) pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retained the capability to increase the interaction with Gab2 and to enhance the activation of the PI3K pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis.

Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced β1 integrin outside-in signaling. In response to β1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.

Identification of cryptotanshinone as an inhibitor of oncogenic protein tyrosine phosphatase SHP2 (PTPN11).

Activating mutations of PTPN11 (encoding the SHP2 phosphatase) are associated with Noonan syndrome, childhood leukemias, and sporadic solid tumors. Virtual screening combined with experimental assays was performed to identify inhibitors of SHP2 from a database of natural products. This effort led to the identification of cryptotanshinone as an inhibitor of SHP2. Cryptotanshinone inhibited SHP2 with an IC50 of 22.50 μM. Fluorescence titration experiments confirmed that it directly bound to SHP2. Enzymatic kinetic analyses showed that cryptotanshinone was a mixed-type and irreversible inhibitor. This drug was further verified for its ability to block SHP2-mediated cell signaling and cellular functions. Furthermore, mouse myeloid progenitors and patient leukemic cells with the activating mutation E76K in PTPN11 were found to be sensitive to this inhibitor. Since cryptotanshinone is used to treat cardiovascular diseases in Asian countries, this drug has a potential to be used directly or to be further developed to treat PTPN11-associated malignancies.

Molecular Targets for the Treatment of Juvenile Myelomonocytic Leukemia

Significant advances in our understanding of the genetic defects and the pathogenesis of juvenile myelomonocytic leukemia (JMML) have been achieved in the last several years. The information gathered tremendously helps us in designing molecular targeted therapies for this otherwise fatal disease. Various approaches are being investigated to target defective pathways/molecules in this disease. However, effective therapy is still lacking. Development of specific target-based drugs for JMML remains a big challenge and represents a promising direction in this field.

Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies

Significance Genetic mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2) are associated with childhood leukemias and solid tumors. The mechanisms by which Ptpn11 mutations induce malignancies are not fully understood. In this study, we demonstrate that Ptpn11 disease mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. This finding reveals a novel mechanism by which deregulation of Shp2 induces malignancies and has important clinical implications. Given that Ptpn11 mutations enhance DNA damage-induced tumorigenesis, caution should be taken when considering DNA-damaging preconditioning for stem cell transplantation therapy or DNA-damaging chemotherapy for Ptpn11-associated malignancies, especially in Noonan syndrome patients with germ-line Ptpn11 mutations, as the risk of therapy-induced malignancies in these patients may be increased. 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.

Factor XII and uPAR upregulate neutrophil functions to influence wound healing

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of &agr;M&bgr;2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine

Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10-25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60-70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.