Dagmar Gotthardt

STAT5 Is a Key Regulator in NK Cells and Acts as a Molecular Switch from Tumor Surveillance to Tumor Promotion

UNLABELLED Natural killer (NK) cells are tightly regulated by the JAK-STAT signaling pathway and cannot survive in the absence of STAT5. We now report that STAT5-deficient NK cells can be rescued by overexpression of BCL2. Our experiments define STAT5 as a master regulator of NK-cell proliferation and lytic functions. Although NK cells are generally responsible for killing tumor cells, the rescued STAT5-deficient NK cells promote tumor formation by producing enhanced levels of the angiogenic factor VEGFA. The importance of VEGFA produced by NK cells was verified by experiments with a conditional knockout of VEGFA in NK cells. We show that STAT5 normally represses the transcription of VEGFA in NK cells, in both mice and humans. These findings reveal that STAT5-directed therapies may have negative effects: In addition to impairing NK-cell-mediated tumor surveillance, they may even promote tumor growth by enhancing angiogenesis. SIGNIFICANCE The importance of the immune system in effective cancer treatment is widely recognized. We show that the new signal interceptors targeting the JAK-STAT5 pathway may have dangerous side effects that must be taken into account in clinical trials: inhibiting JAK-STAT5 has the potential to promote tumor growth by enhancing NK-cell-mediated angiogenesis.

Loss of STAT3 in murine NK cells enhances NK cell–dependent tumor surveillance

The members of the signal transducer and activator of transcription (STAT) family of transcription factors modulate the development and function of natural killer (NK) cells. NK cell-mediated tumor surveillance is particularly important in the bodys defense against hematological malignancies such as leukemia. STAT3 inhibitors are currently being developed, although their potential effects on NK cells are not clear. We have investigated the function of STAT3 in NK cells with Stat3(Δ/Δ)Ncr1-iCreTg mice, whose NK cells lack STAT3. In the absence of STAT3, NK cells develop normally and in normal numbers, but display alterations in the kinetics of interferon-γ (IFN-γ) production. We report that STAT3 directly binds the IFN-γ promoter. In various in vivo models of hematological diseases, loss of STAT3 in NK cells enhances tumor surveillance. The reduced tumor burden is paralleled by increased expression of the activating receptor DNAM-1 and the lytic enzymes perforin and granzyme B. Our findings imply that STAT3 inhibitors will stimulate the cytolytic activity of NK cells against leukemia, thereby providing an additional therapeutic benefit.

Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

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.

STATs in NK-Cells: The Good, the Bad, and the Ugly.

Natural killer (NK)-cells are major players in the fight against viral infections and transformed cells, but there is increasing evidence attributing a disease-promoting role to NK-cells. Cytokines present in the tumor microenvironment shape NK-cell maturation, function, and effector responses. Many cytokines signal via the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway that is also frequently altered and constitutively active in a broad range of tumor cells. As a consequence, there are currently major efforts to develop therapeutic strategies to target this pathway. Therefore, it is of utmost importance to understand the role and contributions of JAK–STAT molecules in NK-cell biology—only this knowledge will allow us to predict effects of JAK–STAT inhibition for NK-cell functions and to successfully apply precision medicine. We will review the current knowledge on the role of JAK–STAT signaling for NK-cell functions and discuss conditions involved in the switch from NK-cell tumor surveillance to disease promotion.

Decreased NK-cell tumour immunosurveillance consequent to JAK inhibition enhances metastasis in breast cancer models

The JAK/STAT pathway is an attractive target for breast cancer therapy due to its frequent activation, and clinical trials evaluating JAK inhibitors (JAKi) in advanced breast cancer are ongoing. Using patient biopsies and preclinical models of breast cancer, we demonstrate that the JAK/STAT pathway is active in metastasis. Unexpectedly, blocking the pathway with JAKi enhances the metastatic burden in experimental and orthotopic models of breast cancer metastasis. We demonstrate that this prometastatic effect is due to the immunosuppressive activity of JAKi with ensuing impairment of NK-cell-mediated anti-tumour immunity. Furthermore, we show that immunostimulation with IL-15 overcomes the enhancing effect of JAKi on metastasis formation. Our findings highlight the importance of evaluating the effect of targeted therapy on the tumour environment. The impact of JAKi on NK cells and the potential value of immunostimulators to overcome the weakened tumour immunosurveillance, are worthwhile considering in the clinical setting of breast cancer.

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.Tumour hypoxia influences both the immune responses and angiogenesis. Here, the authors show that HIF-1α deletion in NK cells impairs NK cytotoxic activity but inhibit tumour growth by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1, thus resulting in non-functional angiogenesis.

In vivo tumor surveillance by NK cells requires TYK2 but not TYK2 kinase activity

Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2−/−) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2−/− mice and mice expressing a kinase-inactive version of TYK2 (Tyk2K923E), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2−/− and Tyk2K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2−/− NK cells (and to a lesser extent of Tyk2K923E NK cells) is impaired. In contrast, the production of interferon γ (IFNγ) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2K923E NK cells against a range of target cells in vitro is higher than that of Tyk2−/− NK cells. Consistently, Tyk2K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2s kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy.

Targeting VEGF-A in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia

Chemotherapy remains a mainstay of cancer treatment but its use is often limited by the development of adverse reactions. Severe loss of body weight (cachexia) is a frequent cause of death in cancer patients and is exacerbated by chemotherapy. We show that genetic inactivation of vascular endothelial growth factor (VEGF)-A in myeloid cells prevents chemotherapy-induced cachexia by inhibiting skeletal muscle loss and the lipolysis of white adipose tissue. It also improves clearance of senescent tumour cells by natural killer cells and inhibits tumour regrowth after chemotherapy. The effects depend on the chemoattractant chemerin, which is released by the tumour endothelium in response to chemotherapy. The findings define chemerin as a critical mediator of the immune response, as well as an important inhibitor of cancer cachexia. Targeting myeloid cell-derived VEGF signalling should impede the lipolysis and weight loss that is frequently associated with chemotherapy, thereby substantially improving the therapeutic outcome.

Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice

Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3Δm) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3Δm mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3Δm CRCs. Moreover, STAT3Δm host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3Δm mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3Δm mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse.

STAT1-S727 - the license to kill

Serine phosphorylation has generally been considered indispensable for full transcriptional activity of STAT proteins. Recent data indicate that CDK8-mediated phosphorylation of signal transducer and activator of transcription 1 (STAT1) on S727 inhibits natural killer (NK) cell cytotoxicity and restrains tumor surveillance. These findings implicate CDK8 as a promising target for immunotherapy.