Aging disrupts circadian gene regulation and function in macrophages.

Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.

CD38 in cancer-associated fibroblasts promotes pro-tumoral activity.

Primary and metastatic melanoma progression are supported by a local microenvironment comprising, inter alia, of cancer-associated fibroblasts (CAFs). We previously reported in orthotropic/syngeneic mouse models that the stromal ectoenzyme CD38 participates in melanoma growth and metastasis. The results presented here suggest that CD38 is a novel regulator of CAFs' pro-tumorigenic functions. Orthotopic co-implantation of CD38 deficient fibroblasts and B16F10 melanoma cells limited tumor size, compared with CD38-expressing fibroblasts. Intrinsically, CAF-CD38 promoted migration of primary fibroblasts toward melanoma cells. Further, in vitro paracrine effects of CAF-CD38 fostered tumor cell migration and invasion as well as endothelial cell tube formation. Mechanistically, we report that CAF-CD38 drives the protein expression of an angiogenic/pro-metastatic signature, which includes VEGF-A, FGF-2, CXCL-12, MMP-9, and HGF. Data suggest that CAF-CD38 fosters tumorigenesis by enabling the production of pro-tumoral factors that promote cell invasion, migration, and angiogenesis.

Stromal CD38 regulates outgrowth of primary melanoma and generation of spontaneous metastasis.

The outgrowth of primary melanoma, the deadliest skin cancer, and generation of metastasis is supported by the tumor microenvironment (TME) which includes non-cancerous cells. Since the TME plays an important role in melanoma pathogenesis, its targeting is a promising therapeutic approach. Thus, it is important to identify proteins in the melanoma TME that may serve as therapeutic targets. Here we show that the nicotinamide adenine dinucleotide glycohydrolase CD38 is a suitable target for this purpose. Loss of CD38 in the TME as well as inhibition of its enzymatic activity restrained outgrowth of primary melanoma generated by two transplantable models of melanoma, B16F10 and Ret-mCherry-sorted (RMS) melanoma cells. Pathological analysis indicated that loss of CD38 increased cell death and reduced the amount of cancer-associated fibroblasts (CAFs) and blood vessels. Importantly, in addition to inhibiting outgrowth of primary melanoma tumors, loss of CD38 also inhibited spontaneous occurrence of RMS pulmonary and brain metastasis. The underlying mechanism may involve, at least in the brain, inhibition of metastasis expansion, since loss of CD38 inhibited the outgrowth of B16F10 and RMS brain tumors that were generated by direct intracranial implantation. Collectively, our results suggest that targeting CD38 in the melanoma TME provides a new therapeutic approach for melanoma treatment.

Incipient Melanoma Brain Metastases Instigate Astrogliosis and Neuroinflammation.

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood-brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359-71. ©2016 AACR.

Exploring the Effects of Glycosylation and Etherification of the Side Chains of the Anticancer Drug Mitoxantrone.

Herein we report the synthesis and biological evaluation of symmetric and asymmetric analogues of the DNA intercalating drug mitoxantrone (MTX) in which the side chains of the parent drug were modified through glycosylation or methyl etherification. Several analogues with glycosylated side chains exhibited higher DNA affinity than the parent MTX. The most potent in vitro cytotoxicity was observed for MTX analogue 8 (1,4-dimethoxy-5,8-bis[2-(2-methoxyethylamino)ethylamino]anthracene-9,10-dione) with methoxy ether containing side chains. Treatment of melanoma-bearing mice with MTX or analogue 8 decreased the intraperitoneal tumor burden relative to untreated mice; the effect of 8 was less pronounced than that of MTX. In vitro metabolism assays of MTX with rabbit liver S9 fraction gave rise to several metabolites; almost no metabolites were detected for MTX analogue 8. The results presented indicate that derivatization of the MTX side chain primary hydroxy groups may result in a significant improvement in DNA affinity and lower susceptibility to the formation of potentially toxic metabolites.

Alzheimer's disease pathology is attenuated in a CD38-deficient mouse model.

OBJECTIVE: Alzheimer's disease (AD)-associated dementia is due to tissue damage caused by amyloid ß (Aß) deposition within the brain and by accompanying neuroinflammation. The nicotinamide adenine dinucleotide (NAD) glycohydrolase CD38, which is expressed by neurons, astrocytes, and microglial cells, regulates inflammatory and repair processes in the brain and other tissues by degrading NAD and repressing the activity of other NAD-consuming enzymes and by producing NAD-derived metabolites that regulate calcium signaling and migration of inflammatory cells. Given the role of CD38 in neuroinflammation and repair, we examined the effect of CD38 deletion on AD pathology. METHODS: We crossed APPswePS1ΔE9 (APP.PS) mice with Cd38(-) (/) (-) mice to generate AD-prone CD38-deficient animals (APP.PS.Cd38(-) (/) (-) ) and examined AD-related phenotypes in both groups. RESULTS: APP.PS.Cd38(-) (/) (-) mice exhibited significant reductions in Aß plaque load and soluble Aß levels compared to APP.PS mice, and this correlated with improved spatial learning. Although CD38 deficiency resulted in decreased microglia/macrophage (MM) accumulation, the transcription profile of the Cd38(-) (/) (-) and Cd38(+/) (+) MM was similar, suggesting that the decreased Aß burden in APP.PS.Cd38(-) (/) (-) mice was not due to alterations in MM activation/function. Instead, APP.PS.Cd38(-) (/) (-) neuronal cultures secreted less Aß and this reduction was mimicked when APP.PS neuronal cultures were treated with inhibitors that blocked CD38 enzyme activity or the signaling pathways controlled by CD38-derived metabolites. Furthermore, ß- and γ-secretase activity was decreased in APP.PS.Cd38(-) (/) (-) mice, which correlated with decreased Aß production. INTERPRETATION: CD38 regulates AD pathology in the APP.PS model of AD, suggesting that CD38 may be a novel target for AD treatment.

Inhibition of glioma progression by a newly discovered CD38 inhibitor.

Glioma, the most common cancer of the central nervous system, has very poor prognosis and no effective treatment. It has been shown that activated microglia/macrophages in the glioma tumor microenvironment support progression. Hence, inhibition of the supporting effect of these cells may constitute a useful therapeutic approach. Recently, using a syngeneic mouse glioma progression model, we showed that the ectoenzyme CD38 regulated microglia activation and, in addition, that the loss of CD38 from the tumor microenvironment attenuated glioma progression and prolonged the life span of the tumor-bearing mice. These studies, which employed wild-type (WT) and Cd38(-/-) C57BL/6J mice, suggest that inhibition of CD38 in glioma microenvironment may be used as a new therapeutic approach to treat glioma. Our study tested this hypothesis. Initially, we found that the natural anthranoid, 4,5-dihydroxyanthraquinone-2-carboxylic acid (rhein), and its highly water-soluble tri-potassium salt form (K-rhein) are inhibitors of CD38 enzymatic (nicotinamide adenine dinucleotide glycohydrolase) activity (IC50 = 1.24 and 0.84 µM, respectively, for recombinant mouse CD38). Treatment of WT, but not Cd38(-/-) microglia with rhein and K-rhein inhibited microglia activation features known to be regulated by CD38 (lipopolysaccharide/IFN-γ-induced activation, induced cell death and NO production). Furthermore, nasal administration of K-rhein into WT, but not Cd38(-/-) C57BL/6J, mice intracranially injected with GL261 cells substantially and significantly inhibited glioma progression. Hence, these results serve as a proof of concept, demonstrating that targeting CD38 at the tumor microenvironment by small-molecule inhibitors of CD38, for example, K-rhein, may serve as a useful therapeutic approach to treat glioma.

CD38 deficiency in the tumor microenvironment attenuates glioma progression and modulates features of tumor-associated microglia/macrophages.

Gliomas are the most frequent primary tumors of the brain, and for highly malignant gliomas there is no successful treatment. The tumor microenvironment contains large numbers of infiltrating microglia and macrophages (MM). There is increasing evidence that the tumor-associated MM support glioma expansion. CD38 is a multifunctional ectoenzyme that uses nicotinamide adenine dinucleotide as a substrate to generate second messengers. Previously we showed that CD38 deficiency modulates microglial "activation" and impaired recovery from head trauma by a microglia-associated mechanism. In view of the supportive role of MM in glioma progression and the role of CD38 in microglia activation, we hypothesize that deficiency of CD38 in the tumor microenvironment would inhibit glioma progression. Using the syngeneic GL261 model of glioma progression in wild-type and CD38 null mice, we show here that CD38 deficiency significantly attenuates glioma expansion and prolongs the life span of the glioma-bearing mice. The CD38 deficiency effect was associated with increased cell death and decreased metalloproteinase-12 expression in the tumor mass, as well as modulation of the tumor-induced MM properties, as indicated by a reduction in the expression of the MM marker F4/80 and matrix metalloproteinases. Our results thus suggest that CD38 participates in the tumor-supporting action of MM and that targeting CD38 might be a potential therapeutic approach for glioma treatment.