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).

We generated a transgenic mouse line that expresses the Cre recombinase under the control of the Ncr1 (p46) promoter. Cre-mediated recombination was tightly restricted to natural killer (NK) cells, as revealed by crossing Ncr1-iCreTg mice to the eGFP-LSLTg reporter strain. Ncr1-iCreTg mice were further used to study NK cell-specific functions of Stat5 (signal transducers and activators of transcription 5) by generating Stat5(f/f) Ncr1-iCreTg animals. Stat5(f/f) Ncr1-iCreTg mice were largely devoid of NK cells in peripheral lymphoid organs. In the bone marrow, NK-cell maturation was abrogated at the NK cell-precursor stage. Moreover, we found that in vitro deletion of Stat5 in interleukin 2-expanded NK cells was incompatible with NK-cell viability. In vivo assays confirmed the complete abrogation of NK cell-mediated tumor control against B16F10-melanoma cells. In contrast, T cell-mediated tumor surveillance against MC38-adenocarcinoma cells was undisturbed. In summary, the results of our study show that STAT5 has a cell-intrinsic role in NK-cell development and that Ncr1-iCreTg mice are a powerful novel tool with which to study NK-cell development, biology, and function.

SummaryIn contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a.

Constitutive activation of STAT5 is critical for the maintenance of chronic myeloid leukemia (CML) characterized by the BCR-ABL oncoprotein. Tyrosine kinase inhibitors (TKIs) for the STAT5-activating kinase JAK2 have been discussed as a treatment option for CML patients. Using murine leukemia models combined with inducible ablation of JAK2, we show JAK2 dependence for initial lymphoid transformation, which is lost once leukemia is established. In contrast, initial myeloid transformation and leukemia maintenance were independent of JAK2. Nevertheless, several JAK2 TKIs induced apoptosis in BCR-ABL(+) cells irrespective of the presence of JAK2. This is caused by the previously unknown direct 'off-target' inhibition of BCR-ABL. Cellular and enzymatic analyses suggest that BCR-ABL phosphorylates STAT5 directly. Our findings suggest uncoupling of the canonical JAK2-STAT5 module upon BCR-ABL expression, thereby making JAK2 targeting dispensable. Thus, attempts to pharmacologically target STAT5 in BCR-ABL(+) diseases need to focus on STAT5 itself.

In BCR-ABL1 ؉ leukemia, drug resistance is often associated with up-regulation of BCR-ABL1 or multidrug transporters as well as BCR-ABL1 mutations. Here we show that the expression level of the transcription factor STAT5 is another parameter that determines the sensitivity of BCR-ABL1 ؉ cells against tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, or dasatinib. Abelson-transformed cells, expressing high levels of STAT5, were found to be significantly less sensitive to TKI-induced apoptosis in vitro and in vivo but not to other cytotoxic drugs, such as hydroxyurea, interferon-␤, or Aca-dC. The STAT5-mediated protection requires tyrosine phosphorylation of STAT5 independent of JAK2 and transcriptional activity. In support of this concept, under imatinib treatment and with disease progression, STAT5 mRNA and protein levels increased in patients with Ph ؉ chronic myeloid leukemia. Based on our data, we propose a model in which disease progression in BCR-ABL1 ؉ leukemia leads to up-regulated STAT5 expression. This may be in part the result of clonal selection of cells with high STAT5 levels. STAT5 then accounts for the resistance against TKIs, thereby explaining the dose escalation frequently required in patients reaching accelerated phase. It also suggests that STAT5 may serve as an attractive target to overcome imatinib resistance in BCR-ABL1 ؉ leukemia. IntroductionMore than 99% of all patients with chronic myeloid leukemia (CML) and approximately 30% of acute lymphoid leukemia are characterized by the t(9;22)(q34;q11) translocation and the Philadelphia chromosome. Two chimeric oncogenic tyrosine kinase products, p185 BCR-ABL1 or p210 BCR-ABL1 , may be generated. 1,2 Whereas p210 BCR-ABL1 is associated with CML, p185 BCR-ABL1 is almost exclusively found in acute lymphoid leukemia. 3 The BCR-ABL1 oncoprotein promotes leukemia development by activating multiple signal transduction pathways that regulate cell proliferation, transformation, and survival. BCR-ABL1-induced acute lymphoid leukemia is characterized by an excess of lymphoblasts and progresses rapidly, whereas BCR-ABL1 ϩ CML is a stem cell-derived disease with distinct phases: chronic phase (CP), which may last for several years, accelerated phase (AP), and blast crisis (BC). 4 Therapy of BCR-ABL1-induced diseases was significantly improved by the development of small molecular weight inhibitors blocking the activity of the ABL1 kinase. Imatinib was the first substance, soon followed by other kinase inhibitors (TKIs), such as dasatinib and nilotinib. [5][6][7] All these TKIs target the enzymatic activity of the ABL1 tyrosine kinases. 6,8,9 Imatinib is now the standard first-line therapy for all CML patients. However, not all CML patients respond equally well. 10 Moreover, approximately 15% to 25% of the patients who initially responded well acquire resistance against imatinib during therapy. The percentage of nonresponders is even higher in AP. [10][11][12] These patients are treated with increased imatinib dosage (600-800 mg/day), secondge...

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs.Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25 þ CML cell line KU812.

In the original Figure 3C, the files from patient B were accidentally uploaded twice for patient B and patient C. The corrected files for patient C have been corrected in the figure below. The authors apologize for any confusion this may have caused the readers.