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.

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...

Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ ABL ؉ human leukemic cells express PI3K␦ and therefore explored its impact on leukemia development. Using PI3K␦-deficient mice, we define a dual role of PI3K␦ in leukemia. We observed a growth-