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Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL+ human leukemic cells express PI3Kδ and therefore explored its impact on leukemia development. Using PI3Kδ-deficient mice, we define a dual role of PI3Kδ 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 PI3Kδ-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kδ accelerated leukemia progression in vivo. However, the absence of PI3Kδ also affected NK cell–mediated tumor surveillance. PI3Kδ-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 PI3Kδ-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 PI3Kδ. Other tumor models confirmed that PI3Kδ-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kδ 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 PI3Kδ inhibitors as antileukemic agents in clinical trials.

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.

The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL–induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression, thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study, we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, because lack of c-JUN impairs proliferation of p185BCR-ABL–transformed cells without affecting their viability. The decreased proliferation of c-JunΔ/Δ cells is associated with the loss of cyclin-dependent kinase 6 (CDK6) expression. In c-JunΔ/Δ cells, CDK6 expression becomes down-regulated upon BCR-ABL–induced transformation, which correlates with CpG island methylation within the 5′ region of Cdk6. We verified the impact of Cdk6 deficiency using Cdk6−/− mice that developed BCR-ABL–induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition, we show that reexpression of CDK6 in BCR-ABL–transformed c-JunΔ/Δ cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the activating protein 1 (AP-1) transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1–regulated DNA methylation on BCR-ABL–induced transformation, thereby accelerating leukemogenesis.

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-β, 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.

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 Stat5f/fNcr1-iCreTg animals. Stat5f/fNcr1-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.

The tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in cancer cells with little effect on normal cells. We have previously shown that TRAIL signaling is altered in most ovarian cancer patients and that resistance to TRAIL contributes to ovarian cancer progression. In this study, we investigated whether resistance to TRAIL may be overcome by a monoclonal TRAILR2 (DR5) agonistic antibody (AD5-10). We found that the joint presence of AD5-10 with TRAIL and natural killer (NK) cells expressing TRAIL resensitizes ovarian cancer cells to apoptosis in vitro and in vivo, respectively. The combination of AD5-10 with carboplatin exerts a more than additive effect in vitro, which may at least partially be explained by the fact that carboplatin triggers DR5 expression on ovarian cancer cells. Moreover, AD5-10 restores the sensitivity of platin-resistant ovarian cancer to carboplatin in vivo. In addition, we found that TRAIL expression and NK cells are abundant in the tumor microenvironment and that depletion of NK cells abolishes the antitumor activity of AD5-10. This indicates that NK-mediated immunosurveillance against ovarian cancer might be mediated by TRAIL and that apoptosis induced by AD5-10 requires the presence of NK cells. In conclusion, this study indicates a key role and strong antitumorigenic effect of DR5 and highlights a novel link between NK-mediated immunosurveillance and activation of DR5-mediated apoptosis in ovarian cancer. Mol Cancer Ther; 9(4); 1007–18. ©2010 AACR.

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.