The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

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-defi cient NK cells can be rescued by overexpression of BCL2. Our experiments defi ne 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-defi cient NK cells promote tumor formation by producing enhanced levels of the angiogenic factor VEGFA. The importance of VEGFA produced by NK cells was verifi ed 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 fi ndings 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. Cancer Discov; 6(4); ©2016 AACR . Ni and Cerwenka, p. 347. See related commentary by INTRODUCTIONNatural killer (NK) cells are innate lymphocytes that develop from a common lymphoid progenitor in the bone marrow ( 1 ). They represent the fi rst line of defense against infected, stressed, and malignant cells. Recent evidence has assigned distinct features and functions to tissue-specifi c NK cells ( 2 ). NK cells have organ-specifi c properties, such as distinct profi les of receptor expression or cytokine production ( 3 ). Uterine NK cells secrete high levels of VEGFA and are involved in placental vascularization. The physiologic functions of other organ-specifi c NK-cell subsets are less well understood ( 4 ). STAT5 Is a Key Regulator in NK Cells and Acts as a Molecular Switch from Tumor Surveillance to Tumor PromotionDagmarAll aspects of NK-cell development are regulated by cytokines, their downstream signaling pathways, and transcriptional regulators. These include key cytokines such as IL2, IL12, IL15, IL18, and IL21 ( 5 ), most of which signal via the common γ chain ( 5 ) and activate the JAK-STAT pathway ( 6 ). JAK kinases (JAK1-3 and TYK2) bind to cytokine receptors and are activated by ligand/receptor binding. The activated kinase phosphorylates STAT transcription factors refs. 6,7 ).Consistent with its function as the major STAT protein downstream of IL7, IL2, and IL15, STAT5 is absolutely essential for conventional NK-cell development and survival; Stat5 Δ / Δ Ncr1-iCre Tg mice lack NK cells ( 8 ). It is also important for lymphoid cell development ( 9 ): STAT5 is constitutively active in a plethora of lymphoid malignancies ( 10 ). Recent studies have described somatic Stat5b mutations as active drivers of lymphoid mali...

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

Tumor-promoting inflammation and avoiding immune destruction are hallmarks of cancer. Here, we demonstrate that the pro-inflammatory cytokine interleukin (IL)-18 is critically involved in these hallmarks in multiple myeloma (MM). Mice deficient for IL-18 were remarkably protected from VkMYC MM progression in a CD8 T cell-dependent manner. The MM-niche-derived IL-18 drove generation of myeloid-derived suppressor cells (MDSCs), leading to accelerated disease progression. A global transcriptome analysis of the immune microenvironment in 73 MM patients strongly supported the negative impact of IL-18-driven MDSCs on T cell responses. Strikingly, high levels of bone marrow plasma IL-18 were associated with poor overall survival in MM patients. Furthermore, our preclinical studies suggested that IL-18 could be a potential therapeutic target in MM.

SummaryThe transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.