Georg Gruenbacher

Novel Aspects of Mevalonate Pathway Inhibitors as Antitumor Agents

The mevalonate pathway for cholesterol biosynthesis and protein prenylation has been implicated in various aspects of tumor development and progression. Certain classes of drugs, such as statins and bisphosphonates, inhibit mevalonate metabolism and therefore have also been tested as antitumor agents. This concept is strongly supported by the recent finding that mutant p53, which is present in more than half of all human cancers, can significantly upregulate mevalonate metabolism and protein prenylation in carcinoma cells. The first evidence that mevalonate pathway inhibitors may have the potential to reverse the malignant phenotype has already been obtained. Moreover, recently discovered immunomodulatory properties of statins and bisphosphonates may also contribute to their known anticancer effects. Drug-induced inhibition of protein prenylation may induce sequential cellular stress responses, including the unfolded protein response and autophagy, that eventually translate into inflammasome-dependent and caspase-1-mediated activation of innate immunity. This review focuses on these novel capabilities of mevalonate pathway inhibitors to beneficially affect tumor biology and contribute to tumor immune surveillance. Clin Cancer Res; 18(13); 3524–31. ©2012 AACR.

DC-like cell-dependent activation of human natural killer cells by the bisphosphonate zoledronic acid is regulated by γδ T lymphocytes

Bisphosphonates are mainly used for the inhibition of osteoclast-mediated bone resorption but also have been shown to induce γδ T-cell activation. Using IL-2-primed cultures of CD56(+) peripheral blood mononuclear cells, we show here that zoledronic acid (zoledronate) could induce IFN-γ production not only in γδ T lymphocytes but, surprisingly, also in natural killer (NK) cells in a manner that depended on antigen-presenting cells, which share properties of inflammatory monocytes and dendritic cells (DCs; here referred to as DC-like cells). In the presence of γδ T lymphocytes, DC-like cells were rapidly eliminated, and NK cell IFN-γ production was silenced. Conversely, in the absence of γδ T lymphocytes, DC-like cells were spared, allowing NK cell IFN-γ production to proceed. γδ T cell-independent NK cell activation in response to zoledronate was because of downstream depletion of endogenous prenyl pyrophosphates and subsequent caspase-1 activation in DC-like cells, which then provide mature IL-18 and IL-1β for the activation of IL-2-primed NK cells. Pharmacologic inhibition of caspase-1 almost abolished IFN-γ production in NK cells and γδ T lymphocytes, indicating that caspase-1-mediated cytokine maturation is the crucial mechanism underlying innate lymphocyte activation in response to zoledronate.

Regulation of mevalonate metabolism in cancer and immune cells

The mevalonate pathway is a highly conserved metabolic cascade and provides isoprenoid building blocks for the biosynthesis of vital cellular products such as cholesterol or prenyl pyrophosphates that serve as substrates for the posttranslational prenylation of numerous proteins. The pathway, which is frequently hyperactive in cancer cells, is considered an important target in cancer therapy, since prenylated members of the Ras superfamily are crucially involved in the control of proliferation, survival, invasion and metastasis of tumour cells. Upstream accumulation and downstream depletion of mevalonate pathway intermediates as induced for instance by aminobisphosphonates translate into different effects in cancer and immune cells. Thus, mevalonate pathway regulation can affect tumour biology either directly or exhibit indirect antitumour effects through stimulating cancer immune surveillance. The present review summarizes major effects of pharmacologic mevalonate pathway regulation in cancer and immune cells that may collaboratively contribute to the efficacy of cancer therapy.

IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells

Statins are inhibitors of cholesterol biosynthesis and protein prenylation that also have been studied in cancer therapy and chemoprevention. With regard to natural killer (NK) cells, only inhibitory effects of statins such as suppression of granule exocytosis have been reported so far. In this study, we show that statins can cooperate with IL-2 to potently induce the activation of CD56(dim) NK cells in a synergistic, time- and dose-dependent fashion. Supplementation experiments revealed that the statin effect was specific to inhibition of their target hydroxymethylglutaryl coenzyme A reductase and that downstream depletion of geranylgeranyl pyrophosphate was responsible for cooperating with IL-2 in NK cell activation. Mechanistic studies revealed that CD56(+)HLA-DR(+)CD14(+) dendritic cell (DC)-like accessory cells mediated the ability of statin to activate NK cells. In contrast, BDCA-1(+) (CD1c(+)) myeloid DCs, which partially expressed CD56, were somewhat less potent. Conventional blood monocytes, which lack CD56, exhibited the lowest accessory cell capacity. NK cell IFN-γ production was IL-12 independent but required endogenous IL-18, IL-1β, and caspase-1 activity. Statins directly induced apoptosis in human cancer cell lines and cooperated with NK cell-derived IFN-γ to generate potent cytotoxic antitumor effects in vitro even in the presence of statin-mediated inhibitory effects on granule exocytosis. Our work reveals novel and unexpected immunomodulatory properties of statins, which might be harnessed for the treatment of cancer.

CD56+ human blood dendritic cells effectively promote TH1-type γδ T-cell responses

CD56+ human dendritic cells (DCs) have recently been shown to differentiate from monocytes in response to GM-CSF and type 1 interferon in vitro. We show here that CD56+ cells freshly isolated from human peripheral blood contain a substantial subset of CD14+CD86+HLA-DR+ cells, which have the appearance of intermediate-sized lymphocytes but spontaneously differentiate into enlarged DC-like cells with substantially increased HLA-DR and CD86 expression or into fully mature CD83+ DCs in response to appropriate cytokines. Stimulation of CD56+ cells containing both DCs and abundant gammadelta T cells with zoledronate and interleukin-2 (IL-2) resulted in the rapid expansion of gammadelta T cells as well as in IFN-gamma, TNF-alpha, and IL-1beta but not in IL-4, IL-10, or IL-17 production. IFN-gamma, TNF-alpha, and IL-1beta production were almost completely abolished by depleting CD14+ cells from the CD56+ subset before stimulation. Likewise, depletion of CD14+ cells dramatically impaired gammadelta T-cell expansion. IFN-gamma production could also be blocked by neutralizing the effects of endogenous IL-1beta and TNF-alpha. Conversely, addition of recombinant IL-1beta, TNF-alpha, or both further enhanced IFN-gamma production and strongly up-regulated IL-6 production. Our data indicate that CD56+ DCs from human blood are capable of stimulating CD56+ gammadelta T cells, which may be harnessed for immunotherapy.

Essential Requirements of Zoledronate-Induced Cytokine and γδ T Cell Proliferative Responses

The potent nitrogen-containing bisphosphonate zoledronate inhibits farnesyl pyrophosphate synthase, a key enzyme of the mevalonate pathway that is often hyperactive in malignant cells. Zoledronate activates human Vγ9Vδ2 T cells, which are immune sentinels of cell stress and tumors, through upstream accumulation of the cognate Ag isopentenyl pyrophosphate. IL-18 was shown to enhance zoledronate-induced γδ T cell activation. Although monocytes have been considered important accessory cells that provide the Ag isopentenyl pyrophosphate, CD56brightCD11c+ NK cells were postulated to mediate the costimulatory effects of IL-18. We report in this article that downstream depletion of geranylgeranyl pyrophosphate (GGPP), which is required for protein prenylation, caused cell stress in monocytes, followed by caspase-1–mediated maturation and release of IL-18, which, in turn, induced γδ T cell CCL2. Likewise, zoledronate caused a substantial delay in γδ T cell expansion, which could be skipped by GGPP supplementation. Moreover, repletion of GGPP, which prevented acute zoledronate toxicity, and supplementation with IL-18, which strongly upregulated IL-2Rα (CD25) and favored the central memory phenotype, were sufficient to enable zoledronate-induced expansion of highly purified γδ T cells, even when starting cell numbers were as low as 104 γδ T cells. Our study reveals essential components of γδ T cell activation and indicates that exogenous IL-18, which can directly costimulate γδ T cells, eliminates the need for any accessory cells. Our findings will facilitate the generation of robust γδ T cells from small blood or tissue samples for cancer immunotherapy and immune-monitoring purposes.

T lymphocyte regulation by mevalonate metabolism.

Metabolites of the mevalonate pathway provide energy, modify critical molecules, and even act as agonists for T cells. T lymphocyte functions, such as cytokine production, appear to require a metabolic shift from oxidative phosphorylation to aerobic glycolysis. Whereas glycolysis and the mitochondrial tricarboxylic acid cycle in lymphocytes are well studied, less attention is paid to the requirements for lipid biosynthesis in the mevalonate pathway. This review, which contains 4 figures and 88 references, discusses how activated T cells rapidly process glucose and funnel acetyl-coenzyme A into mevalonate metabolism and shows that mevalonate metabolites serve quite different purposes in T lymphocyte biology. One important branch of the mevalonate pathway leads to cholesterol, an essential membrane component, and then to the synthesis of steroid hormones, which regulate T cell function. Intermediates of the mevalonate pathway can be attached to proteins and serve as membrane anchors. Finally, several mevalonate metabolites even act as agonists of unconventional T cells, suggesting that immune surveillance of this metabolic pathway occurs. Whereas resting T cells, which have low metabolic requirements, use oxidative phosphorylation (OXPHOS) to maximize their generation of ATP, activated T cells, similar to tumor cells, shift metabolic activity to aerobic glycolysis, which also fuels mevalonate metabolism. Both sterol and nonsterol derivatives of mevalonate affect T cell function. The intracellular availability of sterols, which is dynamically regulated by different classes of transcription factors, represents a metabolic checkpoint that modulates T cell responses. The electron carrier ubiquinone, which is modified with an isoprenoid membrane anchor, plays a pivotal role in OXPHOS, which supports the proliferation of T cells. Isoprenylation also mediates the plasma membrane attachment of the Ras, Rho, and Rab guanosine triphosphatases, which are involved in T cell immunological synapse formation, migration, proliferation, and cytotoxic effector responses. Finally, multiple phosphorylated mevalonate derivatives can act as danger signals for innate-like γδ T cells, thus contributing to the immune surveillance of stress, pathogens, and tumors. We highlight the importance of the mevalonate pathway in the metabolic reprogramming of effector and regulatory T cells.

Mevalonate metabolism in cancer

Cancer cells are characterized by sustained proliferative signaling, insensitivity to growth suppressors and resistance to apoptosis as well as by replicative immortality, the capacity to induce angiogenesis and to perform invasive growth. Additional hallmarks of cancer cells include the reprogramming of energy metabolism as well as the ability to evade immune surveillance. The current review focuses on the metabolic reprogramming of cancer cells and on the immune systems capacity to detect such changes in cancer cell metabolism. Specifically, we focus on mevalonate metabolism, which is a target for drug and immune based cancer treatment.

Stress-related and homeostatic cytokines regulate Vγ9Vδ2 T-cell surveillance of mevalonate metabolism

The potentially oncogenic mevalonate pathway provides building blocks for protein prenylation and induces cell proliferation and as such is an important therapeutic target. Among mevalonate metabolites, only isopentenyl pyrophosphate (IPP) has been considered to be an immunologically relevant antigen for primate-specific, innate-like Vγ9Vδ2 T cells with antitumor potential. We show here that Vγ9Vδ2 T cells pretreated with the stress-related, inflammasome-dependent cytokine interleukin 18 (IL-18) were potently activated not only by IPP but also by all downstream isoprenoid pyrophosphates that exhibit combined features of antigens and cell-extrinsic metabolic cues. Vγ9Vδ2 T cells induced this way effectively proliferated even under severe lymphopenic conditions and the antioxidant N-acetylcysteine significantly improved reconstitution of γδ T cells predominantly with a central memory phenotype. The homeostatic cytokine IL-15 induced the differentiation of effector cells in an antigen-independent fashion, which rapidly produced abundant interferon γ (IFNγ) upon antigen re-encounter. IL-15 induced effector γδ T cells displayed increased levels of the cytotoxic lymphocyte-associated proteins CD56, CD96, CD161 and perforin. In response to stimulation with isoprenoid pyrophosphates, these effector cells upregulated surface expression of CD107a and exhibited strong cytotoxicity against tumor cells in vitro. Our data clarify understanding of innate immunosurveillance mechanisms and will facilitate the controlled generation of robust Vγ9Vδ2 T cell subsets for effective cancer immunotherapy.

Mevalonate metabolism governs cancer immune surveillance

ABSTRACT The metabolic reprogramming that drives immunity engages the mevalonate pathway for cholesterol biosynthesis and protein prenylation. The importance of tight regulation of this metabolic route is reflected by the fact that too low activity impairs cellular function and survival, whereas hyperactivity can lead to malignant transformation. Here, we first address how mevalonate metabolism drives immunity and then highlight ways of the immune system to respond to both, limited and uncontrolled flux through the mevalonate pathway. Immune responses elicited by mevalonate pathway dysregulation may be harnessed to increase the clinical efficacy of current cancer therapy regimens.