IntroductionStat molecules are part of a highly conserved signaling pathway involved in cell-fate decisions like differentiation, proliferation, and apoptosis. [1][2][3] The cytokines interleukin-2, -4, and -7 (IL-2, IL-4, IL-7) regulate important aspects of lymphoid development and are strong activators of the transcription factors Stat5a and Stat5b. 4 The importance of Stat5a/b for lymphoid cells is also underlined by the fact that constitutively activated Stat5a/b are found in several forms of lymphoid leukemia in mice and humans. [5][6][7][8][9][10] Gene knockouts have greatly contributed to our knowledge about Stat transcription factors because they allowed exploration of their physiologic and pathophysiologic functions. 11 So far, all studies investigating the role of Stat5a/b in lymphopoiesis employed gene-targeted mice still expressing a residual protein corresponding to an N-terminal deletion mutant (Stat5a/b⌬N). 4,[12][13][14] Stat5a/b ⌬N/⌬N mice revealed surprisingly mild phenotypes in Band T-cell development and function.Characterization of the lymphoid compartment in Stat5a/b ⌬N/⌬N mice showed a modest reduction of B-and T-lymphoid-cell numbers accompanied by a complete lack of natural killer (NK) cells and CD4 ϩ CD25 ϩ suppressor T cells. 4,13,15 CD8 ϩ T cells were present but failed to respond to ␣-CD3 and IL-2. 4 Mature B-cell numbers in the periphery were also reduced due to an incomplete block at the early pro-B-cell developmental stage (Hardy fraction B). 13,14 Mice lacking IL-7 or the IL-7R have a block at the earliest step of B-cell development at Hardy fraction A and lack mature B-lymphoid cells in the periphery. 16,17 Notably, B-cell development can be rescued in these mice by forced expression of a constitutively active Stat5a/b mutant. 17 In addition, transgenic mice expressing a constitutively active Stat5b (Stat5b-CA) have increased numbers of pro-B cells. 14 As Stat5a/b are critical components in the signaling cascade downstream of IL-7R, abrogation of Stat5a/b was predicted to result in a dramatic phenotype. Thus, the observations in Stat5a/b ⌬N/⌬N mice were difficult to reconcile with the current understanding of signaling pathways controlling B-cell development.Moreover, Stat5a/b transcription factors have been shown to play an important role in various T-cell developmental decisions. Transgenic Stat5b-CA mice display increased numbers of CD8 ϩ but not CD4 ϩ T cells. 18 This implicates Stat5b as an important regulator of CD4 ϩ /CD8 ϩ lineage decision. Moreover, Stat5a/b DNA binding sites were found in regulatory regions of the T-cell receptor ␥ (TCR␥) gene locus, and Stat5b-CA mice displayed a modest increase in ␥␦ T-cell numbers. 18,19 In Stat5a/b ⌬N/⌬N mice, embryonic ␥␦ T-cell development was severely affected, but numbers were rapidly restored after birth. 20 Therefore, the relevance for Stat5a/b in adult ␥␦ thymopoiesis remained elusive. Another finding in Stat5a/b ⌬N/⌬N mice was striking. Among many substrates that are phosphorylated downstream of the Abelson oncogene, Stat5a...

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 G0/G1 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).

Aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases such as tuberculosis or sarcoidosis and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. Here we show that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of Tsc2 was sufficient to induce hypertrophy and proliferation resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 towards increased glycolysis, while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients mTORC1 activation, macrophage proliferation, and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

SUMMARY Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

Persistent activation of Stat5 is frequently found in hematologic neoplasms. Studies conducted with constitutively active Stat5 mutants (Stat51*6 and cS5 F ) have shown that deregulated Stat5 activity promotes leukemogenesis. To investigate the oncogenic properties of these mutants, we used cS5 F -expressing bone marrow cells which induce a multilineage leukemia when transplanted into recipient mice. Here, we show by immunocytochemistry that cS5 F is localized mainly in the cytoplasmic compartment of leukemic cells, suggesting that the transforming nature of cS5 F may be associated with a cytoplasmic function. In support of this hypothesis, we found that cS5 F forms a complex with the p85 subunit of the phosphatidylinositol 3-kinase (PI3-K) and the scaffolding adapter Gab2 in leukemic bone marrow cells, resulting in the activation of Akt/PKB, a crucial downstream target of PI3-K. By using transducible TAT-Gab2 or TAT-Akt recombinant proteins, we were able to demonstrate that activation of the PI3-kinase/Akt pathway by cS5 F molecules through Gab2 is essential for induction of cell growth. We also found that persistently phosphorylated Stat5 in primary cells from patients with myeloid leukemias has a cytoplasmic localization. These data suggest that oncogenic Stat5 proteins exert dual transforming capabilities not only as transcriptional activators but also as cytoplasmic signaling effectors. IntroductionStat5A and Stat5B transcription factors are important mediators of cytokine-induced cell survival and proliferation. 1 There is a large body of evidence indicating that they play crucial roles in hematopoiesis in humans and mice. Stat5a/b Ϫ/Ϫ mice have multiple hematopoietic defects which affect the proliferation and/or survival of both lymphoid and myeloid lineages. [2][3][4][5][6][7] In addition, Stat5 proteins regulate the growth of hematopoietic progenitor cells, and a recent report has suggested that Stat5 might be involved in self-renewal of human CD34 ϩ progenitor cells. 8,9 Deregulation of the Jak-Stat signaling pathway, particularly Stat3 and Stat5, was reported in many different types of cancer, including hematopoietic neoplasms. 10,11 Persistent activation of these transcription factors is frequently found in many tumor cells, most probably as a consequence of deregulated tyrosine kinase activity. Importantly, Stat5 is a common target for different oncoproteins with tyrosine kinase activity such as Tel-Jak2, Bcr-Abl, the mutated forms of Flt3 and c-Kit, and the Jak2V617F mutant which has been recently characterized in various myeloproliferative disorders. [12][13][14][15][16] Furthermore, it was shown that Stat5 plays a critical role in Bcr-Abl-and Tel-Jak2-induced leukemia. [17][18][19] The most direct evidence that constitutive activation of Stat5 is an important causative event in cell transformation came from the analysis of the Stat5 mutants, Stat5A1*6, Stat5B1*6, and cS5 F . These proteins with mutations at residues H 299 3R and S 711/716 3F (Stat5A1*6) or with the single mutation S 711 3F (cS5 ...

The tumor suppressor STAT1 is considered a key regulator of the surveillance of developing tumors. Here, we describe an unexpected tumor-promoting role for STAT1 in leukemia. STAT1 2/2 mice are partially protected from leukemia development, and STAT1 2/2 tumor cells induce leukemia in RAG2 2/2 and immunocompetent mice with increased latency. The low MHC class I protein levels of STAT1 2/2 tumor cells enable efficient NK cell lysis and account for the enhanced tumor clearance. Strikingly, STAT1 2/2 tumor cells acquire increased MHC class I expression upon leukemia progression. These findings define STAT1 as a tumor promoter in leukemia development. Furthermore, we describe the upregulation of MHC class I expression as a general mechanism that allows for the escape of hematopoietic malignancies from immune surveillance.

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in colorectal carcinomas (CRCs). Here, we define the relationship between STAT3 function and the malignant properties of colon carcinoma cells. Elevated activation of STAT3 enhances invasive growth of the CRC cell lines. To address mechanisms through which STAT3 influences invasiveness, the protease mRNA expression pattern of CRC biopsies was analyzed and correlated with the STAT3 activity status. These studies revealed a striking coincidence of STAT3 activation and strong expression of matrix metalloproteinases MMP-1, -3, -7, and -9. Immunohistological examination of CRC tumor specimens showed a clear colocalization of MMP-1 and activated STAT3. Experimentally induced STAT3 activity in CRC cell lines enhanced both the level of MMP-1 mRNA and secreted MMP-1 enzymatic activity. A direct connection of STAT3 activity and transcription from the MMP-1 promoter was shown by reporter gene experiments. Moreover, high-affinity binding of STAT3 to STAT recognition elements in both the MMP-1 and MMP-3 promoter was demonstrated. Xenograft tumors arising from implantation of CRC cells into nude mice showed simultaneous appearance and colocalization of p-Y-STAT3 and MMP-1 expression. Our results link aberrant activity of STAT3 in CRC to malignant tumor progression through upregulated expression of MMPs.

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