Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model.

Introduction: Distinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice. Methods: Plasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination. Results: EVs were significantly increased in the plasma of SLE patients and differentially expressed EV-derived small RNA profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3. Discussion: These data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.

Aromatase-Inhibitor-Induced Musculoskeletal Inflammation Is Observed Independent of Oophorectomy in a Novel Mouse Model.

Aromatase Inhibitors (AIs) block estrogen production and improve survival in patients with hormone-receptor-positive breast cancer. However, half of patients develop aromatase-inhibitor-induced arthralgia (AIIA), which is characterized by inflammation of the joints and the surrounding musculoskeletal tissue. To create a platform for future interventional strategies, our objective was to characterize a novel animal model of AIIA. Female BALB/C-Tg(NFκB-RE-luc)-Xen mice, which have a firefly luciferase NFκB reporter gene, were oophorectomized and treated with an AI (letrozole). Bioluminescent imaging showed significantly enhanced NFκB activation with AI treatment in the hind limbs. Moreover, an analysis of the knee joints and legs via MRI showed enhanced signal detection in the joint space and the surrounding tissue. Surprisingly, the responses observed with AI treatment were independent of oophorectomy, indicating that inflammation is not mediated by physiological estrogen levels. Histopathological and pro-inflammatory cytokine analyses further demonstrated the same trend, as tenosynovitis and musculoskeletal infiltrates were detected in all mice receiving AI, and serum cytokines were significantly upregulated. Human PBMCs treated with letrozole/estrogen combinations did not demonstrate an AI-specific gene expression pattern, suggesting AIIA-mediated pathogenesis through other cell types. Collectively, these data identify an AI-induced stimulation of disease pathology and suggest that AIIA pathogenesis may not be mediated by estrogen deficiency, as previously hypothesized.

Gut dysbiosis is associated with acceleration of lupus nephritis.

The gut microbiota (GM) exerts a strong influence over the host immune system and dysbiosis of this microbial community can affect the clinical phenotype in chronic inflammatory conditions. To explore the role of the GM in lupus nephritis, we colonized NZM2410 mice with Segmented Filamentous Bacteria (SFB). Gut colonization with SFB was associated with worsening glomerulonephritis, glomerular and tubular immune complex deposition and interstitial inflammation compared to NZM2410 mice free of SFB. With SFB colonization mice experienced an increase in small intestinal lamina propria Th17 cells and group 3 innate lymphoid cells (ILC3s). However, although serum IL-17A expression was elevated in these mice, Th17 cells and ILC3s were not detected in the inflammatory infiltrate in the kidney. In contrast, serum and kidney tissue expression of the macrophage chemoattractants MCP-1 and CXCL1 were significantly elevated in SFB colonized mice. Furthermore, kidney infiltrating F4/80+CD206+M2-like macrophages were significantly increased in these mice. Evidence of increased gut permeability or "leakiness" was also detected in SFB colonized mice. Finally, the intestinal microbiome of SFB colonized mice at 15 and 30 weeks of age exhibited dysbiosis when compared to uncolonized mice at the same time points. Both microbial relative abundance as well as biodiversity of colonized mice was found to be altered. Collectively, SFB gut colonization in the NZM2410 mouse exacerbates kidney disease, promotes kidney M2-like macrophage infiltration and overall intestinal microbiota dysbiosis.

In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates.

Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient identification and characterization of candidate substrates are an important first-step to uncovering the function of crosslinked proteins. Herein we describe an optimized and flexible protocol for in vitro TG2 crosslink reactions and substrate incorporation assays. We have previously employed these techniques in the identification of the protein high mobility group box 1 (HMGB1) as a TG2 substrate. However, the protocol can be adapted for identification of any candidate transamidation substrate.

SMAD4 promotes TGF-ß-independent NK cell homeostasis and maturation and antitumor immunity.

SMAD4 is the only common SMAD in TGF-ß signaling that usually impedes immune cell activation in the tumor microenvironment. However, we demonstrated here that selective deletion of Smad4 in NK cells actually led to dramatically reduced tumor cell rejection and augmented tumor cell metastases, reduced murine CMV clearance, as well as impeded NK cell homeostasis and maturation. This was associated with a downregulation of granzyme B (Gzmb), Kit, and Prdm1 in Smad4-deficient NK cells. We further unveiled the mechanism by which SMAD4 promotes Gzmb expression. Gzmb was identified as a direct target of a transcriptional complex formed by SMAD4 and JUNB. A JUNB binding site distinct from that for SMAD4 in the proximal Gzmb promoter was required for transcriptional activation by the SMAD4-JUNB complex. In a Tgfbr2 and Smad4 NK cell-specific double-conditional KO model, SMAD4-mediated events were found to be independent of canonical TGF-ß signaling. Our study identifies and mechanistically characterizes unusual functions and pathways for SMAD4 in governing innate immune responses to cancer and viral infection, as well as NK cell development.

The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens.

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.

IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-κB.

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.

Daily Moderate Exercise Is Beneficial and Social Stress Is Detrimental to Disease Pathology in Murine Lupus Nephritis.

Daily moderate exercise (DME) and stress management are underemphasized in the care of patients with lupus nephritis (LN) due to a poor comprehensive understanding of their potential roles in controlling the inflammatory response. To investigate these effects on murine LN, disease progression was monitored with either DME or social disruption stress (SDR) induction in NZM2410/J mice, which spontaneously develop severe, early-onset LN. SDR of previously established social hierarchies was performed daily for 6 days and DME consisted of treadmill walking (8.5 m/min for 45 min/day). SDR significantly enhanced kidney disease when compared to age-matched, randomly selected control counterparts, as measured by histopathological analysis of H&E staining and immunohistochemistry for complement component 3 (C3) and IgG complex deposition. Conversely, while 88% of non-exercised mice displayed significant renal damage by 43 weeks of age, this was reduced to 45% with exercise. DME also reduced histopathology in kidney tissue and significantly decreased deposits of C3 and IgG complexes. Further examination of renal infiltrates revealed a macrophage-mediated inflammatory response that was significantly induced with SDR and suppressed with DME, which also correlated with expression of inflammatory mediators. Specifically, SDR induced IL-6, TNF-α, IL-1ß, and MCP-1, while DME suppressed IL-6, TNF-α, IL-10, CXCL1, and anti-dsDNA autoantibodies. These data demonstrate that psychological stressors and DME have significant, but opposing effects on the chronic inflammation associated with LN; thus identifying and characterizing stress reduction and a daily regimen of physical activity as potential adjunct therapies to complement pharmacological intervention in the management of autoimmune disorders, including LN.

Thymoquinone exerts potent growth-suppressive activity on leukemia through DNA hypermethylation reversal in leukemia cells.

Thymoquinone (TQ), a bioactive constituent of the volatile oil of Monarda fistulosa and Nigella sativa, possesses cancer-specific growth inhibitory effects, but the underlying molecular mechanisms remain largely elusive. We propose that TQ curbs cancer cell growth through dysfunction of DNA methyltransferase 1 (DNMT1). Molecular docking analysis revealed that TQ might interact with the catalytic pocket of DNMT1 and compete with co-factor SAM/SAH for DNMT1 inhibition. In vitro inhibitory assays showed that TQ decreases DNMT1 methylation activity in a dose-dependent manner with an apparent IC50 of 30 nM. Further, exposure of leukemia cell lines and patient primary cells to TQ resulted in DNMT1 downregulation, mechanistically, through dissociation of Sp1/NFkB complex from DNMT1 promoter. This led to a reduction of DNA methylation, a decrease of colony formation and an increase of cell apoptosis via the activation of caspases. In addition, we developed and validated a sensitive and specific LC-MS/MS method and successfully detected a dynamic change of TQ in mouse plasma after administration of TQ through the tail vein, and determined a tolerable dose of TQ to be 15 mg/kg in mouse. TQ administration into leukemia-bearing mice induced leukemia regression, as indicated by the reversed splenomegaly and the inhibited leukemia cell growth in lungs and livers. Our study for the first time demonstrates that DNMT1-dependent DNA methylation mediates the anticancer actions of TQ, opening a window to develop TQ as a novel DNA hypomethylating agent for leukemia therapy.

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

Liposomal bortezomib is active against chronic myeloid leukemia by disrupting the Sp1-BCR/ABL axis.

The abundance of the BCR/ABL protein critically contributes to CML pathogenesis and drug resistance. However, understanding of molecular mechanisms underlying BCR/ABL gene regulation remains incomplete. While BCR/ABL kinase inhibitors have shown unprecedented efficacy in the clinic, most patients relapse. In this study, we demonstrated that the Sp1 oncogene functions as a positive regulator for BCR/ABL expression. Inactivation of Sp1 by genetic and pharmacological approaches abrogated BCR/ABL expression, leading to suppression of BCR/ABL kinase signaling and CML cell proliferation. Because of potential adverse side effects of bortezomib (BORT) in imatinib-refractory CML patients, we designed a transferrin (Tf)-targeted liposomal formulation (Tf-L-BORT) for BORT delivery. Cellular uptake assays showed that BORT was efficiently delivered into K562 cells, with the highest efficacy obtained in Tf-targeted group. After administered into mice, L-BORT exhibited slower clearance with less toxicity compared to free BORT. Furthermore, L-BORT exposure significantly blocked BCR/ABL kinase activities and sensitized CML cell lines, tumor cells and doxorubicin (DOX) resistant cells to DOX. This occurred through the more pronounced inhibition of BCR/ABL activity by L-BORT and DOX. Collectively, these findings highlight the therapeutic relevance of disrupting BCR/ABL protein expression and strongly support the utilization of L-BORT alone or in combination with DOX to treat CML patients with overexpressing BCR/ABL.

Evaluation of TK1 targeting carboranyl thymidine analogs as potential delivery agents for neutron capture therapy of brain tumors.

In this report we describe studies with N5-2OH, a carboranyl thymidine analog (CTA), which is a substrate for thymidine kinase 1 (TK1), using the F98 rat glioma model. In vivo BNCT studies have demonstrated that intracerebral (i.c.) osmotic pump infusion of N5-2OH yielded survival data equivalent to those obtained with i.v. administration of boronophenylalanine (BPA). The combination of N5-2OH and BPA resulted in a modest increase in MST of F98 glioma bearing rats compared to a statistically significant increase with the RG2 glioma model, as has been previously reported by us (Barth et al., 2008). This had lead us to synthesize a second generation of CTAs that have improved in vitro enzyme kinetics and in vivo tumor uptake (Agarwal et al., 2015).

Epstein-Barr virus-induced gene 3-deficiency leads to impaired antitumor T-cell responses and accelerated tumor growth.

Epstein-Barr virus-induced gene 3 (EBI3) encoded protein can form heterodimers with IL-27P28, and IL-12P35 to form IL-27, and IL-35. However, IL-27 stimulates, whereas IL-35 inhibits antitumor T-cell responses. IL-27 also limits the Foxp3+ regulatory T cell (Treg) population, whereas IL-35 has been shown to expand Tregs and foster Treg suppressive functions. It remains unclear which group of forces are dominant during antitumor T-cell responses. In this study, we evaluated the tumor growth and antitumor T-cell responses in EBI3-deficient mice that lack both IL-27 and IL-35. We found that injecting B16 melanoma cells into EBI3-deficient C57BL/6 mice, or J558 plasmacytoma cells into EBI3-deficient BALB/c mice resulted in significantly increased tumor growth relative to those implanted in wild-type control mice. Tumors from EBI3-deficient mice contained significantly decreased proportions of CD8+ T cells and increased proportions of CD4+FoxP3+ Treg cells as compared to those from EBI3-intact mice. Tumor-infiltrating T cells from EBI3-deficient mice were impaired in their capacity to produce IFNγ. Phenotypically, Tregs from EBI3-deficient mice were highly suppressive and produced IL-10 in the tumor microenvironment. Depletion of Tregs or inactivation of the IL-10 pathway significantly abrogated tumor growth enhancement in Ebi3-/- mice. Finally, we showed that Ebi3-/- mice administered a melanoma vaccine failed to mount a CD8+ T-cell response and the vaccine failed to confer tumor rejection in EBI3-deficient mice. Taken together, these results suggest that Ebi3-/- mice show a phenotype of IL-27-deficiency rather than IL-35-deficiency during anti-tumor T-cell responses. Thus, our results suggest that endogenous IL-27 is critical for both spontaneous and vaccine-induced antitumor T-cell responses.

Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer.

A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogs, designed as substrates and inhibitors of thymidine kinase 1 (TK1) for potential use in boron neutron capture therapy (BNCT) of cancer, was synthesized and evaluated in enzyme kinetic-, enzyme inhibition-, metabolomic-, and biodistribution studies. One of these 2nd generation carboranyl pyrimidine nucleoside analogs (YB18A [3]), having an amino group directly attached to a meta-carborane cage tethered via ethylene spacer to the 3-position of thymidine, was approximately 3-4 times superior as a substrate and inhibitor of hTK1 than N5-2OH (2), a 1st generation carboranyl pyrimidine nucleoside analog. Both 2 and 3 appeared to be 5'-monophosphorylated in TK1(+) RG2 cells, both in vitro and in vivo. Biodistribution studies in rats bearing intracerebral RG2 glioma resulted in selective tumor uptake of 3 with an intratumoral concentration that was approximately 4 times higher than that of 2. The obtained results significantly advance the understanding of the binding interactions between TK1 and carboranyl pyrimidine nucleoside analogs and will profoundly impact future design strategies for these agents.

A chimeric human-mouse model of Sjögren's syndrome.

Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology.

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.

Oral administration of nano-emulsion curcumin in mice suppresses inflammatory-induced NFκB signaling and macrophage migration.

Despite the widespread use of curcumin for centuries in Eastern medicine as an anti-inflammatory agent, its molecular actions and therapeutic viability have only recently been explored. While curcumin does have potential therapeutic efficacy, both solubility and bioavailability must be improved before it can be more successfully translated to clinical care. We have previously reported a novel formulation of nano-emulsion curcumin (NEC) that achieves significantly greater plasma concentrations in mice after oral administration. Here, we confirm the immunosuppressive effects of NEC in vivo and further examine its molecular mechanisms to better understand therapeutic potential. Using transgenic mice harboring an NFκB-luciferase reporter gene, we demonstrate a novel application of this in vivo inflammatory model to test the efficacy of NEC administration by bioluminescent imaging and show that LPS-induced NFκB activity was suppressed with NEC compared to an equivalent amount of curcumin in aqueous suspension. Administration of NEC by oral gavage resulted in a reduction of blood monocytes, decreased levels of both TLR4 and RAGE expression, and inhibited secretion of MCP-1. Mechanistically, curcumin blocked LPS-induced phosphorylation of the p65 subunit of NFκB and IκBα in murine macrophages. In a mouse model of peritonitis, NEC significantly reduced macrophage recruitment, but not T-cell or B-cell levels. In addition, curcumin treatment of monocyte derived cell lines and primary human macrophages in vitro significantly inhibited cell migration. These data demonstrate that curcumin can suppress inflammation by inhibiting macrophage migration via NFκB and MCP-1 inhibition and establish that NEC is an effective therapeutic formulation to increase the bioavailability of curcumin in order to facilitate this response.

The natural product phyllanthusmin C enhances IFN-γ production by human NK cells through upregulation of TLR-mediated NF-κB signaling.

Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.

A nucleolin-DNMT1 regulatory axis in acute myeloid leukemogenesis.

Nucleolin overexpression and DNA hypermethylation have been implicated in cancer pathogenesis, but whether and how these aberrations cooperate in controlling leukemia cell fate remains elusive. Here, we provide the first mechanistic insights into the role of nucleolin in leukemogenesis through creating a DNA hypermethylation profile in leukemia cells. We found that, in leukemia patients, nucleolin levels are significantly elevated and nucleolin overexpression strongly associates with DNMT upregulation and shorter survival. Enforced nucleolin expression augmented leukemia cell proliferation, whereas nucleolin dysfunction by RNA interference and inhibitory molecule AS1411 blocked leukemia cell clonogenic potential in vitro and impaired tumorigenesis in vivo. Mechanistic investigations showed that nucleolin directly activates NFκB signaling, and NFκB activates its downstream effector, DNA methylation machinery. Indeed, nucleolin overexpression increased NFκB phosphorylation and upregulated DNMT1 that is followed by DNA demethylation; by contrast, nucleolin dysfunction dephosphorylated NFκB and abrogated DNMT1 expression, which resulted in decreased global DNA methylation, restored p15INK4B expression and DNA hypomethylation on p15INK4B promoter. Notably, NFκB inactivation diminished, whereas NFκB overexpression enhanced DNMT1 promoter activity and endogenous DNMT1 expression. Collectively, our studies identify nucleolin as an unconventional epigenetic regulator in leukemia cells and demonstrate nucleolin-NFκB-DNMT1 axis as a new molecular pathway underlying AML leukemogenesis.