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T-helper cells express CD4 as a co-receptor that binds to major histocompatibility complex class II to synchronize the immune response against upcoming threats via mediating several cytokines. We have previously reported the presence of CD4 homologues in brown trout. The study of cellular immune responses in brown trout is limited by the availability of specific antibodies. We here describe the generation of a polyclonal antibody against CD4-1 that allows for the investigation of CD4+ cells. We used this novel tool to study CD4+ cells in different tissues during viral haemorrhagic septicaemia infection (VHSV) using flow cytometric technique. Flow cytometric analyses revealed an enhanced level of surface CD4-1 expression in the infected group in major lymphoid organs and in the intestine. These results suggest an important role for the T-helper cells within the immune response against viruses, comparable to the immune response in higher vertebrates.

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.

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.

International audience; Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(Delta N) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants de-creased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis. (Blood. 2010;116(9):1548-1558)

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 G(0)/G(1) 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).

The Stat5 transcription factors Stat5a and Stat5b have been implicated in lymphoid development and transformation. Using the complete Stat5 knockout mice, we have previously shown that Stat5a/bnull/null cells were resistant to transformation and leukemia development induced by Abelson oncogenes, whereas Stat5a/bΔN/ΔN cells readily transformed. So far, these findings showed distinct susceptibility to Abelson-induced transformation of Stat5a/bΔN/ΔN and Stat5a/bnull/null mice and defined Stat5 as key regulator of initial transformation. In this study, we tested whether Stat5a/b is also essential for the maintenance of a transformed state. Therefore we developed a system, where Stat5a/b could be deleted at will. Abelson-transformed B lymphoid cells were generated from Stat5a/bfl/fl gene targeted mice that had been crossed with Mx-Cre transgenic animals. These leukemic Stat5a/bfl/flMxCre cells were then used to test effects of Stat5a/b ablation in vitro and in vivo. In vitro, Stat5a/b deletion resulted in a cell cycle arrest followed by apoptosis. Nine days after deletion, no viable cells could be detected. In line with that, a down-regulation of Stat5 target genes mediating G1/S transition within the cell cycle and viability, such as cyclin D2 and cyclin D3, c-myc and bcl-xL was found. When leukemic Stat5a/bfl/flMxCre cells were injected into wild type or immuno-compromised mice leukemia rapidly developed. Again, deletion of Stat5a/b in vivo within the leukemic cells significantly counteracted disease progression as indicated by an increase of leukemia latency from 16 to 49 days. Eventually, all animals succumbed to a Stat5a/b-positive leukemia indicating that a few residual cells escaped deletion. Moreover, p53 abruption or overexpression of the oncogene did not alter the susceptibility to Stat5 loss of established leukemic cell lines. Taken together our data define a key role for Stat5a/b not only for lymphoid development but also for lymphoid transformation. Stat5a/b is necessary for the initial transformation as well as for leukemia progression. This absolute necessity for the proliferation and viability of Abelson-transformed cells puts Stat5a/b into the spotlight of new therapeutic strategies for the treatment of bcr/abl-induced leukemias.

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.