Bülent Sargin

Translocation Products in Acute Myeloid Leukemia Activate the Wnt Signaling Pathway in Hematopoietic Cells

ABSTRACT The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARα), and PLZF-RARα encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARα, or PLZF-RARα. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (γ-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. β-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

Constitutive Activation of Akt by Flt3 Internal Tandem Duplications Is Necessary for Increased Survival, Proliferation, and Myeloid Transformation

Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, encoding a receptor tyrosine kinase. These mutations induce constitutive tyrosine kinase activity in the absence of the natural Flt3 ligand and confer growth factor independence, increased proliferation, and survival to myeloid precursor cells. The signaling pathways and downstream nuclear targets mediating leukemic transformation are only partly identified. Here, we show that the presence of Flt3-ITD constitutively activates Akt (PKB), a key serine-threonine kinase within the phosphatidylinositol 3-kinase pathway. Constitutive activation of Akt phosphorylated and inhibited the transcription factor Foxo3a. Restored Foxo3a activity reversed Flt3-ITD-mediated growth properties and dominant-negative Akt prevented Flt3-ITD-mediated cytokine independence. Conditional Akt activation targeted to the cell membrane induced cytokine-independent survival, cell cycle progression, and proliferation. Importantly, Akt activation was sufficient to cause in vitro transformation of 32D myeloid progenitor cells and in vivo promoted the development of a leukemia-like myeloid disease. Akt phosphorylation was found in myeloid blasts of 86% of AML patients, suggesting an important role in leukemogenesis. In summary, Akt is necessary for increased survival, proliferation, and leukemic transformation by Flt3-ITD, possibly by inactivation of Foxo transcription factors. These findings indicate that Akt and Foxo transcription factors are attractive targets for therapeutic intervention in AML.

S100A2 Induces Metastasis in Non–Small Cell Lung Cancer

Purpose: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non–small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. Experimental Design: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. Results: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). Conclusions: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Wnt signaling regulates transendothelial migration of monocytes.

The Wnt‐signaling pathway plays a critical role in directing cell fate during embryogenesis. Several lines of evidence also suggest a role in inflammatory processes. Here, we analyzed whether Wnt signaling plays a role in leukocyte inflammatory responses. Monocytes from healthy donors expressed different Frizzled receptors, which are ligands for the Wnt molecules. Activation of the Wnt/β‐catenin pathway by LiCl or Wnt3a increased β‐catenin protein levels in monocytes but not in granulocytes. It is interesting that the activation of Wnt/β‐catenin signaling via Wnt3a in monocytes resulted in a decrease in migration through an endothelial layer (human dermal microvascular endothelial cell‐1). Further experiments revealed that the decrease in transendothelial migration was associated with specific monocyte adherence to endothelial cells after Wnt exposure. The specificity was verified by a lack of Wnt3a‐induced adhesion to fibronectin, laminin, or collagen compared with endothelial interaction. Analysis of the distribution of β‐catenin revealed a Wnt3a‐induced increase of β‐catenin in the cytoplasm. Wnt3a exposure did not result in any activation of the classical Wnt‐target gene c‐myc or a Wnt‐target gene involved in cell adhesion (Connexin43). Our study implicates for the first time a role of canonical Wnt signaling in inflammatory processes in monocytes.

E3 ligase-defective Cbl mutants lead to a generalized mastocytosis and myeloproliferative disease.

Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold. We recently identified the first c-Cbl mutation in human disease in an acute myeloid leukemia patient, called Cbl-R420Q. Here we analyzed the role of Cbl mutants on Kit-mediated transformation. Coexpression of Cbl-R420Q or Cbl-70Z with Kit induced cytokine-independent proliferation, survival, and clonogenic growth. Primary murine bone marrow retrovirally transduced with c-Cbl mutants and transplanted into mice led to a generalized mastocytosis, a myeloproliferative disease, and myeloid leukemia. Overexpression of these Cbl mutants inhibited stem cell factor (SCF)-induced ubiquitination and internalization of Kit. Both Cbl mutants enhanced the basal activation of Akt and prolonged the ligand-dependent activation. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on receptor tyrosine kinases, but independent of their kinase activity. Instead, transformation depends on the Src family kinase Fyn, as c-Cbl coimmunoprecipitated with Fyn and inhibition abolished transformation. These findings may explain primary resistance to tyrosine kinase inhibitors targeted at receptor tyrosine kinases.

Activation of Wnt signaling in cKit-ITD mediated transformation and imatinib sensitivity in acute myeloid leukemia

The Wnt-signaling pathway plays a critical role in directing cell fate during embryogenesis and also in the pathogenesis of cancer. In leukemia, it is well described that activating internal tandem duplications (ITD) mutations in receptor tyrosine kinases like cKit or Flt3 confer to the pathogenesis of cancer. Here, we analyzed whether Wnt-signaling plays a role in cKit-ITD mediated transformation. Stably transfected 32D cells with cKit-ITD cells had higher β-Catenin protein levels compared to the cKit-WT. Analysis of β-Catenin mRNA and protein levels revealed that β-Catenin was regulated at post-transcriptional level in cKit-ITD as well as Flt3-ITD compared to the wildtype. Signaling analyses revealed higher-phosphorylation of GSK3β by oncogenic cKit-ITD. Moreover, activation of Wnt signaling was confirmed by constitutive activation of c-myc luciferase by cKit-ITD cells. Importantly, using dominant negative TCF4, we show that activation of Wnt signaling plays an important role in cKit mediated transformation of myeloid cells. Application of specific receptor tyrosine kinase inhibitors for Flt3 or cKit result in a decrease of β-Catenin that underwent with a decrease of GSK3β phosphorylation, suggesting an indirect mechanism of β-Catenin regulation by oncogenic receptor tyrosine kinases in both ITD mutations. Our study shows the importance of activation of Wnt signaling in leukemia and suggests as attractive target for future therapeutical approaches.

Combination of romiplostim and rituximab: effective therapy of severe immune thrombocytopenia

To the Editor: Immune thrombocytopenia (ITP) leads to accelerated platelet destruction accompanied by reduced platelet production. Here, we describe two patients with severe ITP that were successfully treated with a combination of romiplostim and rituximab. A 59-year-old woman was referred for relapsed ITP. Splenectomy was not possible because of a platelet count of 1000 ⁄lL and signs of bleeding. Further analysis revealed high-titer anti-platelet antibodies in the serum and increased numbers of megakaryocytes in the bone marrow. Initial treatment with prednisolone and intravenous immunoglobulins (IVIG), followed by dexamethasone and anti-rhesus-D immunoglobulin, did not lead to any significant increase in the platelet counts. As the disease was unresponsive to therapy(1), administration of romiplostim was initiated on day 1 (1 lg ⁄kg) with subsequent dose increases (1 lg ⁄kg per week) up to a dose of 6 lg ⁄kg on day 34 after the first dose of romiplostim without any change in platelet counts (Fig. 1). Platelet transfusions as well as administration of dexamethasone, factor XIII, and tranexamic acid were necessary for acute and life-threatening bleeding episodes. Rituximab (375 mg ⁄m) was given on day 40 and day 47 after the first dose of romiplostim, and the last dose of romiplostim (7 lg ⁄kg) was administered on day 41. Platelet counts increased from 3000 ⁄ lL and 6000 ⁄ lL on days 40 and 44 to 239 000 ⁄ lL on day 47. Both romiplostim and rituximab were stopped on day 47, and thromboprophylaxis was started. Platelet counts continued to rise to 1 053 000 ⁄ lL on day 53. Three weeks later, when platelets began to decrease again, the patient decided to undergo splenectomy as definite treatment for ITP. Afterward, platelet counts remained stable (between 500 000 and 600 000 ⁄lL). No further bleeding episodes occurred. Our second patient, a 25-year-old female with a history of two relapses of Hodgkin’s disease was admitted for newly onset petechiae (platelets 4000 ⁄ lL) 1 year after having completed her last chemotherapy and autologous transplant. Direct monoclonal antibody immobilization of platelet antigen (MAIPA) was positive, and bone marrow examination showed an increased megakaryocyte count. Bleeding was initially controlled by IVIG as well as steroids. In light of potential graft failure after multiple previous chemotherapies, an autologous stem cell boost (8.9 · 10 CD34 cells ⁄kg) was given. After more than 4 weeks without response (platelets 4000 ⁄ lL), administration of romiplostim was started (1 lg ⁄kg) (Fig. 1). Seven days later, the patient developed an acute headache. Magnetic resonance tomography of the brain revealed punctual cerebral hemorrhage, and platelet transfusions were administered. Rituximab (375 mg ⁄m weekly) was given on days 12, 19, 26, and 33 after romiplostim treatment initiation. On day 25, platelets increased to 41 000 ⁄ lL and continued to rise to normal levels, and romiplostim doses were not further escalated (4 lg ⁄kg). Interestingly, platelet counts decreased when romiplostim was tapered. ITP patients show a significant decrease in platelet production(2). Romiplostim, a novel thrombopoiesisstimulating protein, effectively binds and activates the human thrombopoietin receptor(3, 4). Clinical trials with romiplostim and eltrombopag(5, 6) have shown sustained elevations of platelet counts in patients with ITP either before or after splenectomy. Previously, romiplostim was combined with corticosteroids, azathioprin, danazol, or other drugs. However, to our knowledge, a combination with rituximab has not been described, although one patient receiving romiplostim 4 weeks after rituximab has been described(3). We reasoned that in cases of ITP unresponsive to both first-line therapy and romiplostim monotherapy, a combination of rituximab and romiplostim may inhibit platelet destruction and at the same time increase platelet production. Indeed, as seen in both cases, this combination can lead to a strong and even overwhelming increase in platelet counts. Frequent laboratory examinations and thromboprophylaxis are therefore necessary to prevent adverse reactions such as thrombotic events. Furthermore, our data reinforce existing recommendations of careful romiplostim dose increases to prevent uncontrolled increases in platelet counts. In the first case, the long latency of platelet increase after romiplostim initiation, the steep increase with rituximab, and the extent of the platelet response to up to 1 000 000 ⁄ lL which is uncommon for rituximab alone(7) strongly suggest that rituximab played a major role in the platelet response. This platelet response to rituximab may be explained by prestimulated thrombopoiesis as a doi:10.1111/j.1600-0609.2009.01406.x European Journal of Haematology 84 (362–364)

Long-Term Effects of the Adenosine Kinase Inhibitor GP-515 on Hepatic Microcirculation Following Hemorrhagic Shock

AbstractBackground: Microcirculatory perfusion failure, known as the “no-reflow phenomenon”, often develops within a few days after ischemia-reperfusion injury and can be a severe problem following resuscitation. Investigations with the adenosine kinase inhibitor GP-515 have shown beneficial protective effects on tissue. We investigated GP-515 in a long-term intravital microscopy rat model of hemorrhagic shock in order to evaluate its potential usage in prevention of multi-organ failure (MOF). Material and Methods: Rats were randomly assigned to one of two shock groups that underwent microscopy after either 2 or 5 days, or to the control group that did not receive shock. After 90 min of hypotension, shock rats received GP-515 (0.25 mg/kg) or saline in a randomized and blinded manner, followed by 3 h of resuscitation. Intravital microscopy was performed after 2 or 5 days. Results: After 2 days, sinusoidal blood flow was significantly improved by GP-515 (40,833 ± 2,499 μm3/s vs. 30,821 ± 3,332 μm3/s; p < 0.05 vs. placebo). Mean sinusoidal diameter was significantly increased in the GP-515 group after 2 days compared with the placebo group (12.08 ± 0.08 μm vs. 10.9 ± 0.08 μm; p < 0.05). After 5 days, the diameters were normalized in all groups. Perfusion index, indicating the proportion of perfused sinusoids to all investigated sinusoids, significantly increased in the GP-515 group (91.5 ± 1.5% compared with the placebo group 83.4 ± 1.8%; p < 0.05) after 2 days. Conclusion: Systemic administration of GP-515 after shock results in a significant improvement of the microcirculation, which might have a long-lasting beneficial effect.