The transcription factors and proto-oncogenes STAT3 and STAT5 are highly activated in hematological malignancies and represent promising therapeutic targets. Whereas the importance of STAT5 as tumor promoter is beyond doubt, the role of STAT3 in hematological cancers is less well understood. Both, enforced as well as attenuated expression of STAT3 were reported in hematopoietic malignancies. Recent evidence implicates STAT3 as key player for tumor immune surveillance as it both mediates the production of and response to inflammatory cytokines. Here we investigated the effects of STAT3 deletion in a BCR/ABL-induced lymphoma model, which is tightly controlled by natural killer (NK) cells in vivo. Upon STAT3 deletion tumor growth is significantly enhanced when compared to STAT3-expressing controls. The increased tumor size upon loss of STAT3 was accompanied by reduced NK cell infiltration and decreased levels of the cytokine IFN-γ and the chemokine RANTES. Upon transplantation into NK cell-deficient mice differences in lymphoma size were abolished indicating that STAT3 expression in the tumor cells controls NK cell-dependent tumor surveillance. Our findings indicate that STAT3 inhibition in lymphoma patients will impair NK cell-mediated tumor surveillance, which needs to be taken into account when testing STAT3 inhibitors in preclinical or clinical trials.

The transcription factor STAT5 (signal transducer and activator of transcription 5) is frequently activated in hematological malignancies and represents an essential signaling node downstream of the BCR-ABL oncogene. STAT5 can be phosphorylated at three positions, on a tyrosine and on the two serines S725 and S779. We have investigated the importance of STAT5 serine phosphorylation for BCR-ABL-induced leukemogenesis. In cultured bone marrow cells, expression of a STAT5 mutant lacking the S725 and S779 phosphorylation sites (STAT5 SASA ) prohibits transformation and induces apoptosis. Accordingly, STAT5 SASA BCR-ABL + cells display a strongly reduced leukemic potential in vivo , predominantly caused by loss of S779 phosphorylation that prevents the nuclear translocation of STAT5. Three distinct lines of evidence indicate that S779 is phosphorylated by group I p21-activated kinase (PAK). We show further that PAK-dependent serine phosphorylation of STAT5 is unaffected by BCR-ABL tyrosine kinase inhibitor treatment. Interfering with STAT5 phosphorylation could thus be a novel therapeutic approach to target BCR-ABL-induced malignancies.

The large difference in phenotypes among tumour populations may stem from the stochastic origin of tumours from distinct cells – tumour cells are assumed to retain the phenotypes of the cells from which they derive. Yet, functional studies addressing the cellular origin of leukaemia are lacking. Here we show that the cells of origin of both, BCR/ABL‐induced chronic myeloid (CML) and B‐cell acute lymphoid leukaemia (B‐ALL), resemble long‐term haematopoietic stem cells (LT‐HSCs). During disease‐maintenance, CML LT‐HSCs persist to function as cancer stem cells (CSCs) that maintain leukaemia and require signalling by the transcription factor STAT5. In contrast, B‐ALL LT‐HSCs differentiate into CSCs that correspond to pro‐B cells. This transition step requires a transient IL‐7 signal and is lost in IL‐7Rα‐deficient cells. Thus, in BCR/ABLp185+ B‐ALL and BCR/ABLp210+ CML, the final phenotype of the tumour as well as the abundance of CSCs is dictated by diverging differentiation fates of their common cells of origin.

Recent findings from our laboratory provide the first indication that overexpression of Bcl-2 in E mu-myc transgenic cells enhances tumor immunosurveillance by inducing NKG2D ligands. Initial evidence suggests that this model is relevant to human patients. Thus, antitumor therapies that target Bcl-2 harbor the risk of reducing tumor immunogenicity.

In Eμ-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the "second hit" affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2(-/-) mice or by Ab-mediated depletion of CD8(+) T or natural killer (NK) cells accelerated formation of BCL-2-overexpressing lymphomas but not of those lacking p53. Most strikingly, enforced expression of BCL-2 prolonged disease latency in the absence of p53, whereas blocking p53 function in BCL-2-overexpressing tumors failed to accelerate disease. This shows that blocking apoptosis in p53-deficient cells by enforcing BCL-2 expression can mitigate disease progression increasing the "immunologic visibility." In vitro cytotoxicity assays confirmed that high expression of BCL-2 protein facilitates NK and T cell-mediated killing. Moreover, we found that high BCL-2 expression is accompanied by significantly increased levels of the NKG2D ligand MULT1, which may account for the enhanced killing. Our findings provide first evidence that the nature of the second hit affects tumor immunosurveillance in c-MYC-driven lymphomas and define a potential shortcoming of antitumor therapies targeting BCL-2.

BACKGROUND & AIMS:Signal transducer and activator of transcription 3 (Stat3) is the main mediator of interleukin-6-type cytokine signaling required for hepatocyte proliferation and hepatoprotection, but its role in sclerosing cholangitis and other cholestatic liver diseases remains unresolved.METHODS:We investigated the role of Stat3 in inflammation-induced cholestatic liver injury and used mice lacking the multidrug resistance gene 2 (mdr2(-/-)) as a model for SC.RESULTS:We show that conditional inactivation of Stat3 in hepatocytes and cholangiocytes (stat3(Deltahc)) of mdr2(-/-) mice strongly aggravated bile acid-induced liver injury and fibrosis. A similar phenotype was observed in mdr2(-/-) mice lacking interleukin-6 production. Biochemical and molecular characterization suggested that Stat3 exerts hepatoprotective functions in both hepatocytes and cholangiocytes. Loss of Stat3 led to increased expression of tumor necrosis factor alpha, which might reduce the barrier function of bile ducts. Moreover, Stat3-deficient hepatocytes displayed up-regulation of bile acid biosynthesis genes and down-regulation of hepatoprotective epidermal growth factor receptor and insulin-like growth factor 1 signaling pathways. Consistently, stat3(Deltahc) mice were more sensitive to cholic acid-induced liver damage than control mice.CONCLUSIONS:Our data suggest that Stat3 prevents cholestasis and liver damage in sclerosing cholangitis via regulation of pivotal functions in hepatocytes and cholangiocytes.

Tumourigenesis caused by the Bcr/Abl oncoprotein is a multi-step process proceeding from initial to tumour-maintaining events and finally results in a complex tumour-supporting network. A key to successful cancer therapy is the identification of critical functional nodes in an oncogenic network required for disease maintenance. So far, the transcription factors Stat3 and Stat5a/b have been implicated in bcr/abl-induced initial transformation. However, to qualify as a potential drug target, a signalling pathway must be required for the maintenance of the leukaemic state. Data on the roles of Stat3 or Stat5a/b in leukaemia maintenance are elusive. Here, we show that both, Stat3 and Stat5 are necessary for initial transformation. However, Stat5- but not Stat3-deletion induces G(0)/G(1) cell cycle arrest and apoptosis of imatinib-sensitive and imatinib-resistant stable leukaemic cells in vitro. Accordingly, Stat5-abrogation led to effective elimination of myeloid and lymphoid leukaemia maintenance in vivo. Hence, we identified Stat5 as a vulnerable point in the oncogenic network downstream of Bcr/Abl representing a case of non-oncogene addiction (NOA).

doi:10.1182/blood-2008-02-139105 Prepublished online Aug 6, 2008;2008 112: 4655-4664€€€€Baumgartner, Peter Valent, Roland P. Piekorz, Michael Freissmuth and Veronika Sexl Eva Eckelhart, Dagmar Stoiber, Eva Weisz, Johannes A. Schmid, Winfried F. Pickl, Christian Eva Zebedin, Olivia Simma, Christian Schuster, Eva Maria Putz, Sabine Fajmann, Wolfgang Warsch,€

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL(+) human leukemic cells express PI3K delta and therefore explored its impact on leukemia development. Using PI3K delta-deficient mice, we define a dual role of PI3K delta in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell-mediated tumor surveillance: Abelson-transformed PI3K delta-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3K delta accelerated leukemia progression in vivo. However, the absence of PI3K delta also affected NK cell-mediated tumor surveillance. PI3K delta-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3K delta-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3K delta. Other tumor models confirmed that PI3K delta-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3K delta in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3K delta inhibitors as antileukemic agents in clinical trials. ( Blood. 2008; 112: 4655-4664)

The process of cancerogenesis is driven by the hierarchical accumulation of genetic changes. So, beside the deregulated expression of an oncogen, additional genetic defects have to take place in order to transform a normal cell into a tumor cell. In case of Burkitt's lymphoma, a c-myc-driven haematological tumor, two predominant so called 2nd hits have been identified. First, loss-of-function mutations in the p19-Mdm2-p53 tumor suppressor pathway, and second, the over-expression of the anti-apoptotic protein Bcl-2. So far it is not known whether these two different 2nd hits translate into cell-autonomous effects or the ability of the immune system of recognizing and destroying the tumor cells. Our studies revealed that neither over-expression of Bcl-2 nor loss-of-function mutations in the p19-Mdm2-p53 tumor suppressor pathway alters any analysed cell-autonomous effect significantly. However, we could specify an impact of these additional genetic lesions on the interaction between immune system and tumor cells. We were able to show that tumor cells over-expressing Bcl-2 can be eliminated by the immune system more effectively than ones with p19-Mdm2-p53 tumor suppressor pathway malfunctions. These data are in line with the analysis regarding 2nd hits of primary tumor samples obtained form an animal model of Burkitt's lymphoma. This analysis showed a clear survival advantage of mice suffering from tumors over-expressing Bcl-2. Taken together, our studies provide basic knowledge concerning the influence of the 2nd hit on the interaction between immune system and tumor cells, which might have prognostic value for immune based cancer therapies.

The Stat5 transcription factors Stat5a and Stat5b have been implicated in lymphoid development and transformation. Using the complete Stat5 knockout mice, we have previously shown that Stat5a/bnull/null cells were resistant to transformation and leukemia development induced by Abelson oncogenes, whereas Stat5a/bΔN/ΔN cells readily transformed. So far, these findings showed distinct susceptibility to Abelson-induced transformation of Stat5a/bΔN/ΔN and Stat5a/bnull/null mice and defined Stat5 as key regulator of initial transformation. In this study, we tested whether Stat5a/b is also essential for the maintenance of a transformed state. Therefore we developed a system, where Stat5a/b could be deleted at will. Abelson-transformed B lymphoid cells were generated from Stat5a/bfl/fl gene targeted mice that had been crossed with Mx-Cre transgenic animals. These leukemic Stat5a/bfl/flMxCre cells were then used to test effects of Stat5a/b ablation in vitro and in vivo. In vitro, Stat5a/b deletion resulted in a cell cycle arrest followed by apoptosis. Nine days after deletion, no viable cells could be detected. In line with that, a down-regulation of Stat5 target genes mediating G1/S transition within the cell cycle and viability, such as cyclin D2 and cyclin D3, c-myc and bcl-xL was found. When leukemic Stat5a/bfl/flMxCre cells were injected into wild type or immuno-compromised mice leukemia rapidly developed. Again, deletion of Stat5a/b in vivo within the leukemic cells significantly counteracted disease progression as indicated by an increase of leukemia latency from 16 to 49 days. Eventually, all animals succumbed to a Stat5a/b-positive leukemia indicating that a few residual cells escaped deletion. Moreover, p53 abruption or overexpression of the oncogene did not alter the susceptibility to Stat5 loss of established leukemic cell lines. Taken together our data define a key role for Stat5a/b not only for lymphoid development but also for lymphoid transformation. Stat5a/b is necessary for the initial transformation as well as for leukemia progression. This absolute necessity for the proliferation and viability of Abelson-transformed cells puts Stat5a/b into the spotlight of new therapeutic strategies for the treatment of bcr/abl-induced leukemias.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family, which transduces cytokine and growth factor signalling. Analysis of TYK2 loss-of-function revealed its important role in immunity to infection, (auto-) immunity and (auto-) inflammation. TYK2-deficient patients unravelled high similarity between mice and men with respect to cellular signalling functions and basic immunology. Genome-wide association studies link TYK2 to several autoimmune and inflammatory diseases as well as carcinogenesis. Due to its cytokine signalling functions TYK2 was found to be essential in tumour surveillance. Lately TYK2 activating mutants and fusion proteins were detected in patients diagnosed with leukaemic diseases suggesting that TYK2 is a potent oncogene. Here we review the cell intrinsic and extrinsic functions of TYK2 in the characteristics preventing and enabling carcinogenesis. In addition we describe an unexpected function of kinase-inactive TYK2 in tumour rejection.

Selective serotonin reuptake inhibitors (SSRIs) have recently been reported to specifically kill malignant cells of B-lymphoid origin, i.e., cells derived from Burkitt lymphoma. Accordingly, SSRIs have been proposed as lead compounds in the development of new approaches to the treatment of lymphoma/leukaemia. Here we attempted to dissect the underlying signaling pathways by comparing susceptible and resistant cell lines. However, we found that all cell lines investigated underwent apoptotic cell death when exposed to SSRI concentrations exceeding 10 μM regardless of whether the cell lines were derived from B- (e.g., Namalwa, Ramos, Daudi, RL7), T-lymphoid tumors (e.g., Molt-4, Jurkat, CCRF-CEM) or other sources. The structure–activity relationship readily distinguished the pro-apoptotic and growth inhibitory effect of SSRIs from their eponymous action (blockage of the serotonin transporter): acetylation of the SSRIs fluvoxamine and paroxetine abrogated the ability of these compounds to inhibit 5HT-uptake, but did not impair their cytotoxic action. Based on these data we conclude that (i) SSRIs inhibit growth of transformed cells, but that (ii) this effect is neither specific for malignant cells nor specific for any particular cellular subset. (iii) The pro-apoptotic effect of SSRIs (at μM concentrations) is unrelated to their principal pharmacological action, i.e., inhibition of serotonin uptake (at nM concentrations). SSRIs or improved versions thereof are therefore unlikely to represent useful lead compounds for inducing apoptosis in B-cell derived tumors: the underlying mechanism is not confined to any specific cell lineage.

The PI3Kδ isoform is a candidate drug target in leukemia. Here, we explored its role in Abelson-induced leukemia. Frank leukemia emerges if the tumor cells have managed to outwit the immune system. The absence of PI3Kδ affected both the tumor cells and the NK cells. Abelsontransformed PI3Kδ-/cells induced leukemia in RAG2-/animals with a significantly increased latency, implicating PI3Kδ in tumor progression. NK cell function, however, was also contingent on PI3Kδ. PI3Kδ-/NK cells failed to lyse target cells. Capacitance measurements revealed the underlying defect: in PI3Kδ-/NK cells lytic granules did not fuse with the cell membrane. Accordingly, transplanted leukemic cells killed PI3Kδ-/animals more rapidly, both in syngeneic (PI3Kδ-/-) or immunocompromised (RAG2-/PI3Kδ-/-) animals. Our observations define a dual function of PI3Kδ in leukemia and document that the action of PI3Kδ in the NK compartment is as relevant to the survival of the mice as the delayed tumor progression. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

The blood group-related Lewis Y antigen is expressed on the majority of human cancers of epithelial origin with only limited expression on normal tissue. Therefore, the Lewis Y antigen represents an interesting candidate for antibody-based treatment strategies. Previous experiments showed that the humanized Lewis Y-specific monoclonal antibody, IGN311, reduced ErbB-receptor-mediated stimulation of mitogen-activated protein kinase by altering receptor recycling. Here, we tested whether binding of IGN311 to growth factor receptors is relevant also to inhibition of tumor growth in vivo. Prolonged incubation with IGN311 of human tumor cell lines, which express high levels of ErbB1 (A431) or ErbB2 (SK-BR-3), resulted in down-regulation of the receptors and inhibition of cell proliferation. IGN311 inhibited the growth of tumors derived from A431 cells xenografted in nude mice. Treatment with IGN311 was associated with a down-regulation of ErbB1 in the excised tumor tissue. Importantly, these effects of IGN311 were also mimicked by the Fab fragment of IGN311. These data indicate that tumor cell growth inhibition by IGN311 cannot solely be accounted for by invoking cellular and humoral immunological mechanisms. A direct effect on signaling via binding to Lewis Y glycosylated growth factor receptors on tumor cells is also likely to contribute to the therapeutic effect of IGN311 in vivo.