SOCS1 cooperates with FLT3-ITD in the development of myeloproliferative disease by promoting the escape from external cytokine control.

Activating mutations in the receptor tyrosine kinase FLT3 are frequently found in acute myelogenous leukemia patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control that regulates normal hematopoiesis. We have recently demonstrated that FLT3-internal tandem duplication (ITD), when localized to the biosynthetic compartment, aberrantly activates STAT5. Here, we show that one of the target genes induced by STAT5 is suppressor of cytokine signaling (SOCS)1-a surprising finding for a known tumor suppressor. Although SOCS1 expression in murine bone marrow severely impaired cytokine-induced colony growth, it failed to inhibit FLT3-ITD-supported colony growth, indicating resistance of FLT3-ITD to SOCS1. In addition, SOCS1 coexpression did not affect FLT3-ITD-mediated signaling or proliferation. Importantly, SOCS1 coexpression inhibited interferon-α and interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-mediated growth inhibitory effects. In a murine bone marrow transplantation model, the coexpression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared with FLT3-ITD alone (P < .01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting leukemogenesis. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

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.

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.

Activation of Wnt signalling in acute myeloid leukemia by induction of Frizzled-4.

Wnt signalling regulates proliferation, self renewal and cell fate. Aberrant Wnt signalling is thought to contribute to AML pathogenesis by enhancing self renewal. Herein, we provide evidence for increased expression of Frizzled-4, a receptor for Wnt ligands, in primary AML blasts compared to normal bone marrow on the protein level. In addition, Frizzled-4 is highly expressed in human CD34 positive cells as well as in lineage negative sorted mouse bone marrow cells. Functionally, Frizzled-4 expression modulates apoptosis and enhances Wnt3a induced beta-catenin stability in myeloid progenitor cells. Frizzled-4-dependent beta-catenin stabilization is dkk-1 sensitive, implicating a specific Wnt-ligand/Frizzled-receptor interaction. These findings indicate enhanced sensitivity of AML blasts for Wnt-ligands and suggest an additional mechanism of Wnt signalling activation in the pathogenesis of AML.

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 beta-Catenin protein levels compared to the cKit-WT. Analysis of beta-Catenin mRNA and protein levels revealed that beta-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 GSK3beta 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 beta-Catenin that underwent with a decrease of GSK3beta phosphorylation, suggesting an indirect mechanism of beta-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.

Adjuvant therapy with small hairpin RNA interference prevents non-small cell lung cancer metastasis development in mice.

Development of distant metastasis is the major reason for cancer-related deaths worldwide. Adjuvant therapy approaches after local therapies are most effective when specific targets are inhibited. Recently, we identified S100P overexpression as a strong predictor for metastasis development in early-stage non-small cell lung cancer (NSCLC) patients. Here, we show that S100P overexpression increased angiogenesis in and metastasis formation from s.c. xenotransplants of NSCLC cells. Plasmid-derived short hairpin RNAs (shRNA) were developed as specific adjuvant therapy. I.v. injected shRNA against S100P significantly decreased S100P protein expression in xenograft tumors and inhibited tumor angiogenesis in vivo. Metastasis formation 8 weeks after primary tumor resection was significantly reduced. Lung metastases developed in 31% of mice treated with S100P-targeting shRNAs compared with 64% in control shRNA-treated mice (P < 0.05). These findings suggest that RNA interference-based therapy approaches can be highly effective in the adjuvant setting.

CDDO induces granulocytic differentiation of myeloid leukemic blasts through translational up-regulation of p42 CCAAT enhancer binding protein alpha.

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of tumor cells in vitro and in vivo. Here we assessed the effects of CDDO on CCAAT enhancer-binding protein alpha (CEBPA), a transcription factor critical for granulocytic differentiation. In HL60 acute myeloid leukemia (AML) cells, CDDO (0.01 to 2 muM) induces apoptosis in a dose-dependent manner. Conversely, subapoptotic doses of CDDO promote phagocytic activity and granulocytic-monocytic differentiation of HL60 cells through increased de novo synthesis of p42 CEBPA protein. CEBPA translational up-regulation is required for CDDO-induced granulocytic differentiation and depends on the integrity of the CEBPA upstream open reading frame (uORF). Moreover, CDDO increases the ratio of transcriptionally active p42 and the inactive p30 CEBPA isoform, which, in turn, leads to transcriptional activation of CEBPA-regulated genes (eg, GSCFR) and is associated with dephosphorylation of eIF2alpha and phosphorylation of eIF4E. In concordance with these results, CDDO induces a CEBPA ratio change and differentiation of primary blasts from patients with acute myeloid leukemia (AML). Because AML is characterized by arrested differentiation, our data suggest the inclusion of CDDO in the therapy of AML characterized by dysfunctional CEBPA expression.

Flt3-dependent transformation by inactivating c-Cbl mutations in AML.

In acute myeloid leukemia (AML), mutational activation of the receptor tyrosine kinase (RTK) Flt3 is frequently involved in leukemic transformation. However, little is known about a possible role of highly expressed wild-type Flt3 in AML. The proto-oncogene c-Cbl is an important regulator of RTK signaling, acting through its ubiquitin ligase activity and as a platform for several signaling adaptor molecules. Here, we analyzed the role of c-Cbl in Flt3 signal transduction and myeloid transformation. C-Cbl physically interacted with Flt3 and was tyrosine phosphorylated in the presence of Flt3-ligand (FL). Overexpression of a dominant-negative form of c-Cbl (Cbl-70Z) inhibited FL-induced Flt3 ubiquitylation and internalization, indicating involvement of c-Cbl in Flt3 signaling. DNA sequencing of AML bone marrow revealed a case with a c-Cbl point mutation (Cbl-R420Q). Cbl-R420Q inhibited Flt3 internalization and ubiquitylation. Coexpression of Cbl-R420Q or Cbl-70Z with Flt3 induced cytokine-independent growth and survival of 32Dcl3 cells in the absence of FL. Also, the mutant Cbl proteins altered the amplitude and duration of Flt3-dependent signaling events. Our results indicate an important role of Cbl proteins in Flt3 signal modulation. Also, the data suggest a novel mechanism of leukemic transformation in AML by mutational inactivation of negative RTK regulators.

Activation mechanisms of STAT5 by oncogenic Flt3-ITD.

Mutations in the receptor tyrosine kinase Flt3 represent a very common genetic lesion in acute myeloid leukemia (AML). Internal tandem duplication (ITD) mutations clustered in the juxtamembrane domain are the most frequent and best characterized mutations found in Flt3. Oncogenic activation of Flt3 by ITD mutations is known to activate aberrant signaling including activation of STAT5 and repression of myeloid transcription factors Pu.1 and c/EBP-alpha. However, the mechanisms of STAT5 activation by Flt3-ITD remain unclear. Using small molecule inhibitors and cell lines deficient for Src family kinases or Jak2 or Tyk2, here we show that Flt3-ITD-induced STAT5 activation is independent of Src or Jak kinases. Also, overexpression of SOCS1, an inhibitor of Jak kinases, inhibited IL-3- but not Flt3-ITD-mediated STAT5 activation. Furthermore, in vitro kinase assays revealed that STAT5 is a direct target of Flt3. Taken together, our data provide the mechanistic basis of STAT5 activation by Flt3-ITD.

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/beta-catenin pathway by LiCl or Wnt3a increased beta-catenin protein levels in monocytes but not in granulocytes. It is interesting that the activation of Wnt/beta-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 beta-catenin revealed a Wnt3a-induced increase of beta-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.

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.

AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations.

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein alpha (c/EBPalpha) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D cells in suspension cultures. However, in contrast to Flt3-ITD and similar to wild-type Flt3 (Flt3-WT), Flt3-TKD cannot support colony formation in semisolid media. Also, in contrast to Flt3-ITD, neither Flt3-WT nor Flt3-TKD induced activation or induction of STAT5 target genes. Flt3-TKD also failed to repress c/EBPalpha and Pu.1. No significant differences were observed in receptor autophosphorylation and the phosphorylation of Erk-1 and -2, Akt, and Shc. Importantly, TKD but not ITD mutations were a log power more sensitive toward the tyrosine kinase inhibitor protein kinase C 412 (PKC412) than Flt3-WT. In conclusion, Flt3-ITD and Flt3-TKD mutations display differences in their signaling properties that could have important implications for their transforming capacity and for the design of mutation-specific therapeutic approaches.

Flt3 tandem duplication mutations cooperate with Wnt signaling in leukemic signal transduction.

Activating Flt3 mutations occur in about 30% of patients with acute myeloid leukemia (AML), often as in-frame internal tandem duplication (ITD) at the juxtamembrane domain of the receptor. These mutations transform hematopoietic cell lines and primary mouse bone marrow. Here, we analyzed the interaction between oncogenic Flt3-ITD mutations and the Wingless-type (Wnt) signaling pathway in the myeloid progenitor cell line 32D. Microarray analyses revealed higher mRNA expression of Frizzled-4, a receptor for Wnt ligands in 32D/Flt3-ITD cells. Findings were verified by quantitative realtime reverse transcription-polymerase chain reaction (RT-PCR) and on the protein level. Compared with 32D/Flt3-WT (wild-type) cells, 32D/Flt3-ITD cells also showed greatly enhanced beta-catenin protein levels, irrespective of their exposure to Wnt3a, a ligand inducing the canonical Wnt signal transduction pathway. In addition, 5 of 7 AML samples with Flt3-ITD mutations expressed high beta-catenin protein levels, whereas patients with wild-type Flt3 did not. Also, Flt3-ITD induced enhanced T-cell factor (TCF)-dependent transcriptional activity and the induction of the Wnt target gene c-myc. In the presence of Flt3-WT or Flt3-ITD signaling, Wnt3a slightly increased 32D cell proliferation. However, transfection experiments with dominant-negative (dn) TCF4 revealed a strong dependence of Flt3-ITD-mediated clonogenic growth on TCF activity. Taken together, our results indicate that Flt3-ITD and Wnt-dependent signaling pathways synergize in myeloid transformation.

RGS2 is an important target gene of Flt3-ITD mutations in AML and functions in myeloid differentiation and leukemic transformation.

Activating fetal liver tyrosine kinase 3 (Flt3) mutations represent the most common genetic aberrations in acute myeloid leukemia (AML). Most commonly, they occur as internal tandem duplications in the juxtamembrane domain (Flt3-ITD) that transform myeloid cells in vitro and in vivo and that induce aberrant signaling and biologic functions. We identified RGS2, a regulator of G-protein signaling, as a gene specifically repressed by Flt3-ITD. Here we demonstrate an important role of RGS2 in Flt3-ITD-mediated transformation. RGS2 was repressed after forced expression of activating Flt3 mutations in 2 myeloid cell lines (32Dcl3 and NB4). Furthermore, RGS2 was repressed in Flt3-mutation-positive AML cases in comparison to Flt3-mutation-negative cases, especially in Flt3-ITD-positive cases with a high ITD-to-wild-type (WT) ratio. Coexpression of RGS2 with Flt3-ITD inhibited Flt3-ITD-induced autonomous proliferation and clonal growth of 32D cells. RGS2 also inhibited Flt3-ITD-induced phosphorylation of Akt and glycogen synthase kinase beta (Gsk3-beta) without influencing signal transducer and activator of transcription 5 (STAT5) activation. In addition, RGS2 reinduced the expression of Flt3-ITD-repressed CCAAT/enhancer-binding protein alpha (c/EBPalpha) and antagonized the Flt3-ITD-induced differentiation block in 32D cells. Expression analyses in myeloid cell lines revealed induction of RGS2 during granulocytic but not during monocytic differentiation. Taken together, RGS2 is a novel mediator of myeloid differentiation, and its repression is an important event in Flt3-ITD-induced transformation.

Translocation products in acute myeloid leukemia activate the Wnt signaling pathway in hematopoietic cells.

The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha 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-RARalpha, or PLZF-RARalpha. 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 (gamma-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. beta-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.

Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations.

The receptor tyrosine kinase Flt3 is expressed and functionally important in early myeloid progenitor cells and in the majority of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor occur in 25% of AML cases. Previously, we have shown that these mutations activate the receptor and induce leukemic transformation. In this study, we performed genome-wide parallel expression analyses of 32Dcl3 cells stably transfected with either wild-type or 3 different ITD isoforms of Flt3. Comparison of microarray expression analyses revealed that 767 of 6586 genes differed in expression between FLT3-WT- and FLT3-ITD-expressing cell lines. The target genes of mutationally activated Flt3 resembled more closely those of the interleukin 3 (IL-3) receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-2 was up-regulated on the mRNA and the protein level in Flt3-ITD-expressing cells. Further experiments indicated that Pim-2 function was important for clonal growth of 32D cells. Several genes repressed by the mutations were found to be involved in myeloid gene regulation. Pu.1 and C/EBPalpha, both induced by ligand-activation of wild-type Flt3, were suppressed in their expression and function by the Flt3 mutations. In conclusion, internal tandem duplication mutations of Flt3 activate transcriptional programs that partially mimic IL-3 activity. Interestingly, other parts of the transcriptional program involve novel, IL-3-independent pathways that antagonize differentiation-inducing effects of wild-type Flt3. The identification of the transcriptional program induced by ITD mutations should ease the development of specific therapies.