Requirement for CDK6 in MLL-rearranged acute myeloid leukemia.

Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts.

GITR ligand provided by thrombopoietic cells inhibits NK cell antitumor activity.

Thrombocytopenia inhibits tumor growth and especially metastasis in mice, whereas additional depletion of NK cells reverts this antimetastatic phenotype. It has therefore been speculated that platelets may protect hematogenously disseminating tumor cells from NK-dependent antitumor immunity. Tumor cells do not travel through the blood alone, but are rapidly coated by platelets, and this phenomenon has been proposed to shield disseminating tumor cells from NK-mediated lysis. However, the underlying mechanisms remain largely unclear. In this study, we show that megakaryocytes acquire expression of the TNF family member glucocorticoid-induced TNF-related ligand (GITRL) during differentiation, resulting in GITRL expression by platelets. Upon platelet activation, GITRL is upregulated on the platelet surface in parallel with the α-granular activation marker P-selectin. GITRL is also rapidly mobilized to the platelet surface following interaction with tumor cells, which results in platelet coating. Whereas GITRL, in the fashion of several other TNF family members, is capable of transducing reverse signals, no influence on platelet activation and function was observed upon GITRL triggering. However, platelet coating of tumor cells inhibited NK cell cytotoxicity and IFN-γ production that could partially be restored by blocking GITR on NK cells, thus indicating that platelet-derived GITRL mediates NK-inhibitory forward signaling via GITR. These data identify conferment of GITRL pseudoexpression to tumor cells by platelets as a mechanism by which platelets may alter tumor cell immunogenicity. Our data thus provide further evidence for the involvement of platelets in facilitating evasion of tumor cells from NK cell immune surveillance.

The BCR/ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity.

In chronic myeloid leukemia (CML), BCR/ABL-mediated oncogenic signaling can be targeted with the BCR/ABL-inhibitors Imatinib, Nilotinib and Dasatinib. However, these agents may also affect anti-tumor immunity. Here, we analyzed the effects of the 3 BCR/ABL-inhibitors on natural killer (NK) cell reactivity. Exposure of CML cells (K562, Meg-01) to pharmacological concentrations of Imatinib, Nilotinib and Dasatinib diminished expression of ligands for the activating immunoreceptor NKG2D to a similar extent. This resulted in comparably reduced NK cell cytotoxicity and IFN-gamma production. When direct effects on NK cell responses to K562 and primary CML cells as well as activating cytokines were studied, Dasatinib was found to abrogate NK cytotoxicity and cytokine production. Nilotinib did not alter cytotoxicity but, at high levels, impaired NK cytokine production, while Imatinib had no direct influence on NK cell reactivity. Of note, Nilotinib, but not the other BCR/ABL-inhibitors increased cell death within the preferentially cytokine-secreting CD56(bright)CD16(-) NK cell subset, which may, at least in part, serve to explain the effect of Nilotinib on NK cytokine production. Analysis of NK cell signaling revealed that Dasatinib inhibited proximal signaling events leading to decreased phosphorylation of PI3K and ERK that are crucial for NK cell reactivity. Imatinib and Nilotinib, in contrast, showed no relevant effect on NK cell PI3K or ERK activity. In light of the potential role of NK cells in the immunesurveillance of residual leukemia and for future combinatory immunotherapeutic approaches, our data indicate that choice and dosing of the most suitable BCR/ABL-inhibitor for a given patient require careful consideration.

Glucocorticoid-induced TNFR-related (GITR) protein and its ligand in antitumor immunity: functional role and therapeutic modulation.

The ability of the tumor necrosis factor receptor (TNFR) family member GITR to modulate immune responses has been the subject of multiple studies. Initially thought to be critically involved in governing functions of regulatory T cells, GITR and its ligand GITRL have meanwhile been found to modulate the reactivity of various different cell types and to influence a broad variety of immunological conditions including the immune response against tumors. Not only GITR, but also GITRL is capable of transducing signals, and the consequences of GITR-GITRL interaction may vary among different effector cell types, differ upon signal transduction via the receptor, the ligand, or both, depend on the level of an ongoing immune response, and even differ among mice and men. In this paper, we address available data on GITR and its ligand in immune responses and discuss the role and potential therapeutic modulation of this molecule system in antitumor immunity.

The wolf in sheep's clothing: Platelet-derived "pseudo self" impairs cancer cell "missing self" recognition by NK cells.

Metastasis is strongly inhibited in thrombocytopenic mice. This phenotype is reversed by NK cell depletion which indicates that platelets may facilitate tumor progression and metastasis by interfering with NK cell immunosurveillance. Understanding the underlying mechanisms may help us to reinforce anti-tumor immunity and NK-based immunotherapy in cancer patients.

Platelet-derived MHC class I confers a pseudonormal phenotype to cancer cells that subverts the antitumor reactivity of natural killer immune cells.

Natural killer (NK) cells are cytotoxic lymphocytes that play an important role in tumor immunosurveillance, preferentially eliminating targets with low or absent expression of MHC class I and stress-induced expression of ligands for activating NK receptors. Platelets promote metastasis by protecting disseminating tumor cells from NK cell immunosurveillance, but the underlying mechanisms are not well understood. In this study, we show that tumor cells rapidly get coated in the presence of platelets in vitro, and circulating tumor cells of cancer patients display coexpression of platelet markers. Flow cytometry, immunofluorescent staining, confocal microscopy, and analyses on an ultrastructural level using immunoelectron microscopy revealed that such coating may cause transfer of MHC class I onto the tumor cell surface resulting in high-level expression of platelet-derived normal MHC class I. The resulting "phenotype of false pretenses" disrupts recognition of tumor cell missing self, thereby impairing cytotoxicity and IFN-γ production by NK cells. Thus, our data indicate that platelets, by conferring an unsuspicious "pseudonormal" phenotype, may enable a molecular mimicry that allows metastasizing tumor cells to downregulate MHC class I, to escape T-cell-mediated immunity without inducing susceptibility to NK cell reactivity.

Modulation of natural killer cell anti-tumor reactivity by platelets.

Natural killer (NK) cells may prevent tumor progression and metastasis. Apart from the direct interaction with their targets, NK cell activity is influenced by the reciprocal interplay with other hematopoietic cells. While the interaction of NK cells e.g. with dendritic cells or monocytes/macrophages is well characterized, knowledge regarding their crosstalk with platelets, another central component of the blood, is still fragmentary. However, studies in mice and men clearly document a strong dependence of tumor progression and metastasis on quantitatively and functionally normal platelets. In mice, metastasis is inhibited by thrombocytopenia, and this effect is reversed by additional NK cell depletion, indicating that platelets may 'indirectly' contribute to tumor dissemination by impairing NK cell anti-tumor reactivity. In humans, circumstantial evidence indicates that metastasizing malignant cells do not travel through the blood alone, but efficiently attract and get coated by platelets, thereby causing release of platelet granule content. Beyond this secretion of various growth factors and cytokines/chemokines, platelets may also influence NK cell function by immunoregulatory molecules expressed on the platelet surface. Here, we review the available data regarding tumor-platelet-NK cell interaction focusing on metastatic tumor spread and discuss the molecular mechanisms underlying this trilateral crosstalk.

Platelet-derived transforming growth factor-beta down-regulates NKG2D thereby inhibiting natural killer cell antitumor reactivity.

Natural killer (NK) cells play an important role in cancer immunosurveillance and may prevent tumor progression and metastasis due to their ability to mediate direct cellular cytotoxicity and by releasing immunoregulatory cytokines, which shape adaptive immune responses. Their reactivity is governed by various activating and inhibitory molecules expressed on target cells and reciprocal interactions with other hematopoietic cells such as dendritic cells. In mice, thrombocytopenia inhibits metastasis, and this is reversed by NK cell depletion, suggesting that platelets are an important additional player in NK cell-tumor interaction. Moreover, it has been shown that metastasizing tumor cells do not travel through the blood alone but are rapidly coated by platelets. However, the knowledge about the molecular mechanisms by which platelets influence NK cells is fragmentary at best. Here we show that platelet-derived soluble factors, secreted on coating of tumor cells or after stimulation with classic platelet agonists, impair NK cell antitumor reactivity resulting in diminished granule mobilization, cytotoxicity, and IFN-gamma production. The impaired NK cell reactivity was not due to induction of apoptosis but mediated by down-regulation of the activating immunoreceptor natural killer group 2, member D (NKG2D) on NK cells by platelet-derived transforming growth factor beta (TGF-beta). Neutralization of TGF-beta in platelet releasate not only prevented NKG2D down-regulation but also restored NK cell antitumor reactivity. Thus, our data elucidate the molecular basis of the previously described influence of platelets on NK cell antitumor reactivity and suggest that therapeutic intervention in tumor cell-platelet interaction and the resulting TGF-beta release by platelets may serve to enhance antitumor immunity.