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).

IntroductionMajor progress has been achieved in the past decade in understanding the biology of B-cell chronic lymphocytic leukemia (CLL), leading to new therapeutic concepts and a trend toward improved survival. However, the disease remains incurable, and many patients develop drug resistance. [1][2][3] Despite the long lifespan of CLL cells in vivo, these cells undergo rapid and spontaneous apoptosis in vitro but can be rescued by marrow stromal cells, [4][5][6] nurse-like cells, 7 and follicular dendritic cells. 8 There are also indications that the stromal cells mediate resistance to chemotherapy in CLL cells. 9 This point to the dependence of CLL cells on antiapoptotic stimuli that could be provided in vivo by marrow microenvironment 10,11 and may have a major effect on disease progression and response or resistance to therapy.Bone marrow microenvironment is a complex structure that comprises accessory cells (stromal cells, adipocytes, reticulum cells, endothelial cells, follicular dendritic cells, T cells, and macrophages), matrix proteins, and soluble factors, including growth factors and cytokines. 11,12 Bone marrow stromal cells (BMSCs) represent a major component of the marrow microenvironment. They originate from the mesenchymal stem cells and have hematopoietic supportive properties. They also produce matrix proteins, express integrin ligands, and release several cytokines and growth factors that are involved in the generation and maturation of normal and leukemic B cells. [11][12][13] Therefore, BMSCs could provide a suitable milieu for the development and survival of CLL cells. However, the antiapoptotic cascades and the molecular events that are activated upon the interaction between CLL cells and BMSCs are not fully identified.Several stimuli that are endogenously produced in the microenvironment were shown to activate the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. These include cytokines and growth factors, 14 adhesion molecules and matrix proteins, 15 and involve receptors that are expressed on the surface of CLL cells, including the B-cell receptor, CD19, and CD5. [16][17][18] This strongly suggests that the PI3-K/Akt pathway might play a central role in the interaction between CLL cells and the bone marrow microenvironment. The PI3-K/Akt cascade contributes to the regulation of many cellular processes, including motility, proliferation, apoptosis, and tumorigenesis. 14,19 Class I of PI3-K family is the best characterized and comprises p110␣, p110␤, p110␥, and The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From p110␦ isotypes. 14,19 The first generation of pan-PI3-K inhibitors (LY294002 and wortmannin) provided substantial information on the molecular mechanism of action and ...

Oncogenic tyrosine kinases (TK) usually convert growth factor-dependent cells to factor independence with autonomous proliferation. However, TK-driven neoplasms often are indolent and characterized by cell differentiation rather than proliferation. A prototype of an indolent TK-driven neoplasm is indolent systemic mastocytosis. We found that the D816V-mutated variant of KIT, a TK detectable in most patients with systemic mastocytosis, induces cluster formation and expression of several mast cell differentiation and adhesion Ags, including microphthalmia transcription factor, IL-4 receptor, histamine, CD63, and ICAM-1 in IL-3-dependent BaF3 cells. By contrast, wild-type KIT did not induce cluster formation or mast cell differentiation Ags. Additionally, KIT D816V, but not wild-type KIT, induced STAT5 activation in BaF3 cells. However, despite these intriguing effects, KIT D816V did not convert BaF3 cells to factor-independent proliferation. Correspondingly, BaF3 cells with conditional expression of KIT D816V did not form tumors in nude mice. Together, the biologic effects of KIT D816V in BaF3 cells match strikingly with the clinical course of indolent systemic mastocytosis and with our recently established transgenic mouse model, in which KIT D816V induces indolent mast cell accumulations but usually does not induce a malignant mast cell disease. Based on all these results, it is hypothesized that KIT D816V as a single hit may be sufficient to cause indolent systemic mastocytosis, whereas additional defects may be required to induce aggressive mast cell disorders.

IntroductionHematopoietic development is regulated by cytokine-or growth factor-activated signaling pathways, among which the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway plays a major role. [1][2][3] Four Jak kinases and 7 Stat proteins regulate a wide spectrum of cellular functions such as proliferation, survival, and differentiation. 4 Stats are latent transcription factors that constantly shuttle between the nucleus and the cytoplasm. Their activity is tightly regulated by protein tyrosine kinases. Phosphorylation of a positionally conserved tyrosine residue triggers dimerization, efficient nuclear translocation, and subsequent binding of Stats to specific promoter sequences. 5 Target genes regulated by Stat5 proteins and their cofactors are essential for hematopoietic stem cell maintenance, lineage commitment, self-renewal, and survival of committed hematopoietic progenitors as well as for mature cells of both myeloerythroid and lymphoid lineage. [6][7][8] Although there is considerable functional overlap, Stat5a and Stat5b proteins also have distinct functions due to isoformspecific differences in mRNA levels, 3,5 nucleocytoplasmic shuttling, 9,10 and activation by tyrosine and/or serine phosphorylation. 11 Hyperactivated Stat5a and Stat5b proteins have been implicated in several hematopoietic malignancies and many solid tumors. [1][2][3] Yet to date, there are no reports of activating mutations in Stat5 proteins. Persistent tyrosine phosphorylation of Stat5 was found to be caused by deregulated cytokine signaling 12 or perturbation of upstream molecules, which mostly results from receptor mutations or chromosomal rearrangement. Well-known examples of this are the point mutation Jak2-V617F 13,14 and chromosomal translocations such as Bcr-Abl. 15,16 Furthermore, it has been shown that the presence of Stat5 proteins is required for Bcr-Abl-induced transformation and development of leukemia. 17 This is why Stat5 proteins and their activation status are thought to have a key role in leukemogenesis. Moreover, the transforming capacity of oncogenic Stat5 is not limited to its role as a transcription factor in the nucleus. Recent studies have demonstrated that oncogenic Stat5a also has a cytoplasmic role that links Jak/Stat signaling to the activation of the PI3K-Akt-mTOR signaling pathway via Gab2 [18][19][20] and that Stat5 acts as a repressor of Bcl-2 member-regulating microRNAs (miRNAs) 15/16. 21 In addition to tyrosine phosphorylation, the activity of several Stat proteins was shown to be modulated by serine phosphorylation (reviewed in Decker and Kovarik 22 ). In the highly homologous Stat5a and Stat5b proteins, distinct serine residues in the carboxyterminal transactivation domain, the least conserved region of Stat proteins, 2,3 were found to be phosphorylated. These serine moieties, however, are located in perfectly conserved Pro-Ser-Pro (PSP) motifs at positions 725 (Stat5a) and 730 (Stat5b 11,[23][24][25][26][27] Using a mouse transplantation model, 28,29 the pr...

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In E-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 (

Stat transcription factors have been implicated in tumorigenesis in mice and men. Stat3 and Stat5 are considered powerful proto-oncogenes, whereas Stat1 has been demonstrated to suppress tumor formation. We demonstrate here for the first time that a constitutive active version of Stat3alpha (Stat3alphaC) may also suppress transformation. Mouse embryonic fibroblasts (MEFs) deficient for p53 can be transformed with either c-myc or with rasV12 alone. Interestingly, transformation by c-myc is efficiently suppressed by co-expression of Stat3alphaC, but Stat3alphaC does not interfere with transformation by the rasV12-oncogene. In contrast, transplantation of bone marrow cells expressing Stat3alphaC induces the formation of a highly aggressive T cell leukemia in mice. The leukemic cells invaded multiple organs including lung, heart, salivary glands, liver and kidney. Interestingly, transplanted mice developed a similar leukemia when the bone marrow cells were transduced with Stat3beta, which is also constitutively active when expressed at significant levels. Our experiments demonstrate that Stat3 has both - tumor suppressing and tumor promoting properties.