ZEB1 shapes AML immunological niches, suppressing CD8 T cell activity while fostering Th17 cell expansion.

Acute myeloid leukemia (AML) progression is influenced by immune suppression induced by leukemia cells. ZEB1, a critical transcription factor in epithelial-to-mesenchymal transition, demonstrates immune regulatory functions in AML. Silencing ZEB1 in leukemic cells reduces engraftment and extramedullary disease in immune-competent mice, activating CD8 T lymphocytes and limiting Th17 cell expansion. ZEB1 in AML cells directly promotes Th17 cell development that, in turn, creates a self-sustaining loop and a pro-invasive phenotype, favoring transforming growth factor ß (TGF-ß), interleukin-23 (IL-23), and SOCS2 gene transcription. In bone marrow biopsies from AML patients, immunohistochemistry shows a direct correlation between ZEB1 and Th17. Also, the analysis of ZEB1 expression in larger datasets identifies two distinct AML groups, ZEB1high and ZEB1low, each with specific immunological and molecular traits. ZEB1high patients exhibit increased IL-17, SOCS2, and TGF-ß pathways and a negative association with overall survival. This unveils ZEB1's dual role in AML, entwining pro-tumoral and immune regulatory capacities in AML blasts.

Getting everyone to agree on gene signatures for murine macrophage polarization in vitro.

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4).

Diet and Exercise in the Management of Polycystic Ovary Syndrome: Practical Considerations for Person-Centered Care.

Polycystic ovary syndrome (PCOS) is a complex multisystem condition associated with life-long reproductive, metabolic, and psychological symptoms. Individuals with PCOS are at an increased risk of cardiovascular disease and type 2 diabetes, with approximately 70% of all PCOS cases presenting with insulin resistance. Lifestyle interventions have historically been recommended as first-line therapies for the management of PCOS-related cardiometabolic disorders. The term "lifestyle management" incorporates a multifaceted approach to dietary, exercise, and behavioral strategies, aiming to promote a healthy lifestyle. This approach has been commonly employed in practice, in particular through exercise and dietary modulation, due to its effect on cardiometabolic outcomes as well as its tolerability. Furthermore, there is evidence to suggest that combining dietary change with exercise may yield the greatest improvements in clinical outcomes. However, such practices require careful consideration and coordination, as there are instances where certain exercise and/or dietary prescriptions may compromise the effectiveness of the respective interventions. Thus, this review aims to provide practical guidance on diet and exercise planning in the routine care of PCOS. Such recommendations include emphasizing realistic and achievable goals, as well as minimizing barriers to lifestyle changes in order to increase the long-term sustainability of this treatment strategy.

Correction to "An Unexpected Deuterium-Induced Metabolic Switch in Doxophylline".

[This corrects the article DOI: 10.1021/acsmedchemlett.2c00166.].

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

NAMPT and NAPRT serum levels predict response to anti-TNF therapy in inflammatory bowel disease.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyl transferase (NAPRT) are key intracellular enzymes that participate in the biosynthesis on NAD but have also been shown to be released as proinflammatory cytokines. A number of reports have shown that circulating NAMPT is increased in serum of patients with inflammatory disorders, including inflammatory bowel diseases (IBD), while nothing is known regarding circulating NAPRT and the presence of both cytokines in IBD patient stools. In the present study, we evaluated eNAMPT and eNAPRT levels in a large cohort of IBD patients not on biological therapy and in a subset that then was prescribed biologics. Methods: We conducted a retro-perspective study on 180 patients, of which 111 underwent subsequent biological treatment (adalimumab, vedolizumab, and ustekinumab). We analyzed eNAMPT and eNAPRT concentrations in serum and faces of IBD patients, correlating them with response to biologics. Results: We now report that eNAMPT and eNAPRT are significantly increased in both serum and stools of IBD patients. NAMPT and NAPRT levels correlate with disease severity, with C reactive protein and with serum IL-6 levels. Importantly, levels of NAMPT in patients starting treatment with adalimumab correlate with response failure at three months: patients with levels above 4 ng/ml were significantly less likely to obtain benefit. Serum NAMPT as a biomarker of response yields a sensitivity of 91% and a specificity of 100%. Conclusion: The present work strongly suggests that a prospective trial evaluating eNAMPT and eNAPRT levels in relation to response to biologicals in IBD should be initiated.

The Predictive Role of Extracellular NAPRT for the Detection of Advanced Fibrosis in Biopsy-Proven Non-Alcoholic Fatty Liver Disease.

Intrahepatic oxidative stress is a key driver of inflammation and fibrogenesis in non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of extracellular Nicotinamide phosphoribosyltransferase (eNAMPT) and extracellular nicotinic acid phosphoribosyltransferase (eNAPRT) for the detection of advanced fibrosis. eNAMPT and eNAPRT were tested in 180 consecutive biopsy-proven NAFLD patients and compared with liver stiffness (LS) and the FIB-4 score. eNAMPT was similarly distributed across fibrosis stages, whereas eNAPRT was increased in patients with advanced fibrosis (p = 0.036) and was associated with advanced fibrosis (OR 1.08, p = 0.016). A multiple stepwise logistic regression model containing significant variables for advanced fibrosis (eNAPRT, type 2 diabetes, age, male sex, ALT) had an area under the curve (AUC) of 0.82 (Se 89.6%, Sp 67.3%, PPV 46.7%, NPV 93.8%) when compared to that of LS (0.79; Se 63.5%, Sp 86.2%, PPV 66.0%, NPV 84.8%) and to that of the FIB-4 score (0.73; Se 80.0%, Sp 56.8%, PPV 44.9%, NPV 86.6%). The use of eNAPRT in clinical practice might allow for the better characterization of NAFLD patients at higher risk of disease progression.

Identification of novel aza-analogs of TN-16 as disrupters of microtubule dynamics through a multicomponent reaction.

Despite novel biological targets emerging at an impressive rate for anticancer therapy, antitubulin drugs remain the backbone of numerous oncological protocols and their efficacy has been demonstrated in a wide variety of adult and pediatric cancers. In the present contribution, we set to develop analogs of a potent but neglected antitubulin agent, TN-16, originally discovered via modification of tenuazonic acid (3-acetyl-5-sec-butyltetramic acid). To this extent, we developed a novel multicomponent reaction to prepare TN-16, and then we applied the same reaction for the synthesis of aza-analogs. In brief, we prepared a library of 62 novel compounds, and three of these retained nanomolar potencies. TN-16 and the active analogs are cytotoxic on cancer cell lines and, as expected from antitubulin agents, induce G2/M cell cycle arrest. These agents lead to a disruption of the microtubules and an increase in α-tubulin acetylation and affect in vitro polymerization, although they have a lesser effect in cellular tubulin polymerization assays.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

An Unexpected Deuterium-Induced Metabolic Switch in Doxophylline.

Precision deuteration has become part of the medicinal chemist's toolbox, but its usefulness can be undermined by unpredictable metabolic switch effects. Herein we report the deuteration of doxophylline, a drug used in the treatment of asthma and COPD that undergoes extensive oxidative metabolism. Labeling of the main metabolic soft spots triggered an unexpected multidirectional metabolic switch that, while not improving the pharmacokinetic parameters, changed the metabolic scenario and, in turn, the pharmacodynamic features in two murine models of lung injury.

Extracellular nicotinamide phosphoribosyltransferase boosts IFNγ-induced macrophage polarization independently of TLR4.

Nicotinamide phosphoribosyltransferase (NAMPT), alongside being a crucial enzyme in NAD synthesis, has been shown to be a secreted protein (eNAMPT), whose levels are increased in patients affected by immune-mediated disorders. Accordingly, preclinical studies have highlighted that eNAMPT participates in the pathogenesis of several inflammatory diseases. Herein, we analyzed the effects of eNAMPT on macrophage-driven inflammation. RNAseq analysis of peritoneal macrophages (PECs) demonstrates that eNAMPT triggers an M1-skewed transcriptional program, and this effect is not dependent on the enzymatic activity. Noteworthy, both in PECs and in human monocyte-derived macrophages, eNAMPT selectively boosts IFNγ-driven transcriptional activation via STAT1/3 phosphorylation. Importantly, the secretion of eNAMPT promotes the chemotactic recruitment of myeloid cells, therefore providing a potential positive feedback loop to foster inflammation. Last, we report that these events are independent of the activation of TLR4, the only eNAMPT receptor that has hitherto been recognized, prompting the knowledge that other receptors are involved.

Insight Into Nicotinamide Adenine Dinucleotide Homeostasis as a Targetable Metabolic Pathway in Colorectal Cancer.

Tumour cells modify their cellular metabolism with the aim to sustain uncontrolled proliferation. Cancer cells necessitate adequate amounts of NAD and NADPH to support several enzymes that are usually overexpressed and/or overactivated. Nicotinamide adenine dinucleotide (NAD) is an essential cofactor and substrate of several NAD-consuming enzymes, such as PARPs and sirtuins, while NADPH is important in the regulation of the redox status in cells. The present review explores the rationale for targeting the key enzymes that maintain the cellular NAD/NADPH pool in colorectal cancer and the enzymes that consume or use NADP(H).

A Selective Competitive Inhibitor of Aldehyde Dehydrogenase 1A3 Hinders Cancer Cell Growth, Invasiveness and Stemness In Vitro.

Aldehyde dehydrogenase 1A3 (ALDH1A3) belongs to an enzymatic superfamily composed by 19 different isoforms, with a scavenger role, involved in the oxidation of a plethora of aldehydes to the respective carboxylic acids, through a NAD+-dependent reaction. Previous clinical studies highlighted the high expression of ALDH1A3 in cancer stem cells (CSCs) correlated to a higher risk of cancer relapses, chemoresistance and a poor clinical outcome. We report on the structural, biochemical, and cellular characterization of NR6, a new selective ALDH1A3 inhibitor derived from an already published ALDH non-selective inhibitor with cytotoxic activity on glioblastoma and colorectal cancer cells. Crystal structure, through X-Ray analysis, showed that NR6 binds a non-conserved tyrosine residue of ALDH1A3 which drives the selectivity towards this isoform, as supported by computational binding simulations. Moreover, NR6 shows anti-metastatic activity in wound healing and invasion assays and induces the downregulation of cancer stem cell markers. Overall, our work confirms the role of ALDH1A3 as an important target in glioblastoma and colorectal cells and propose NR6 as a promising molecule for future preclinical studies.

Prostaglandin E2 and Cancer: Insight into Tumor Progression and Immunity.

The involvement of inflammation in cancer progression has been the subject of research for many years. Inflammatory milieu and immune response are associated with cancer progression and recurrence. In different types of tumors, growth and metastatic phenotype characterized by the epithelial mesenchymal transition (EMT) process, stemness, and angiogenesis, are increasingly associated with intrinsic or extrinsic inflammation. Among the inflammatory mediators, prostaglandin E2 (PGE2) supports epithelial tumor aggressiveness by several mechanisms, including growth promotion, escape from apoptosis, transactivation of tyrosine kinase growth factor receptors, and induction of angiogenesis. Moreover, PGE2 is an important player in the tumor microenvironment, where it suppresses antitumor immunity and regulates tumor immune evasion, leading to increased tumoral progression. In this review, we describe the current knowledge on the pro-tumoral activity of PGE2 focusing on its role in cancer progression and in the regulation of the tumor microenvironment.

Recent Advances in NAMPT Inhibitors: A Novel Immunotherapic Strategy.

Nicotinamide adenine dinucleotide (NAD) is a cofactor of many enzymatic reactions as well as being a substrate for a number of NAD-consuming enzymes (e.g., PARPS, sirtuins, etc). NAD can be synthesized de novo starting from tryptophan, nicotinamide, nicotinic acid, or nicotinamide riboside from the diet. On the other hand, the nicotinamide that is liberated by NAD-consuming enzymes can be salvaged to re-form NAD. In this former instance, nicotinamide phosphoribosyltransferase (NAMPT) is the bottleneck enzyme. In the many cells in which the salvage pathway is predominant, NAMPT, therefore, represents an important controller of intracellular NAD concentrations, and as a consequence of energy metabolism. It is, therefore, not surprising that NAMPT is over expressed by tumoral cells, which take advantage from this to sustain growth rate and tumor progression. This has led to the initiation of numerous medicinal chemistry programs to develop NAMPT inhibitors in the context of oncology. More recently, however, it has been shown that NAMPT inhibitors do not solely target the tumor but also have an effect on the immune system. To add complexity, this enzyme can also be secreted by cells, and in the extracellular space it acts as a cytokine mainly through the activation of Toll like Receptor 4 (TLR4), although it has not been clarified yet if this is the only receptor responsible for its actions. While specific small molecules have been developed only against the intracellular form of NAMPT, growing evidences sustain the possibility to target the extracellular form. In this contribution, the most recent evidences on the medicinal chemistry of NAMPT will be reviewed, together with the key elements that sustain the hypothesis of NAMPT targeting and the drawbacks so far encountered.

Progress in the Field of Aldehyde Dehydrogenase Inhibitors: Novel Imidazo[1,2-a]pyridines against the 1A Family.

Members of the aldehyde dehydrogenase 1A family are commonly acknowledged as hallmarks of cancer stem cells, and their overexpression is significantly associated with poor prognosis in different types of malignancies. Accordingly, treatments targeting these enzymes may represent a successful strategy to fight cancer. In this work we describe a novel series of imidazo[1,2-a]pyridines, designed as aldehyde dehydrogenase inhibitors by means of a structure-based optimization of a previously developed lead. The novel compounds were evaluated in vitro for their activity and selectivity against the three isoforms of the ALDH1A family and investigated through crystallization and modeling studies for their ability to interact with the catalytic site of the 1A3 isoform. Compound 3f emerged as the first in class submicromolar competitive inhibitor of the target enzyme.

Neutralization of extracellular NAMPT (nicotinamide phosphoribosyltransferase) ameliorates experimental murine colitis.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is increased in inflammatory bowel disease (IBD) patients, and its serum levels correlate with a worse prognosis. In the present manuscript, we show that eNAMPT serum levels are increased in IBD patients that fail to respond to anti-TNFα therapy (infliximab or adalimumab) and that its levels drop in patients that are responsive to these therapies, with values comparable with healthy subjects. Furthermore, eNAMPT administration in dinitrobenzene sulfonic acid (DNBS)-treated mice exacerbates the symptoms of colitis, suggesting a causative role of this protein in IBD. To determine the druggability of this cytokine, we developed a novel monoclonal antibody (C269) that neutralizes in vitro the cytokine-like action of eNAMPT and that reduces its serum levels in rodents. Of note, this newly generated antibody is able to significantly reduce acute and chronic colitis in both DNBS- and dextran sulfate sodium (DSS)-induced colitis. Importantly, C269 ameliorates the symptoms by reducing pro-inflammatory cytokines. Specifically, in the lamina propria, a reduced number of inflammatory monocytes, neutrophils, Th1, and cytotoxic T lymphocytes are found upon C269 treatment. Our data demonstrate that eNAMPT participates in IBD and, more importantly, that eNAMPT-neutralizing antibodies are endowed with a therapeutic potential in IBD. KEY MESSAGES: What are the new findings? Higher serum eNAMPT levels in IBD patients might decrease response to anti-TNF therapy. The cytokine-like activity of eNAMPT may be neutralized with a monoclonal antibody. Neutralization of eNAMPT ameliorates acute and chronic experimental colitis. Neutralization of eNAMPT limits the expression of IBD inflammatory signature. Neutralization of eNAMPT impairs immune cell infiltration in lamina propria.

Discovery of Highly Potent Benzimidazole Derivatives as Indoleamine 2,3-Dioxygenase-1 (IDO1) Inhibitors: From Structure-Based Virtual Screening to in Vivo Pharmacodynamic Activity.

In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical, and biological investigations led to the identification of a novel class of IDO1 inhibitors based on a benzimidazole substructure. This family of compounds is endowed with an extensive bonding network in the protein active site, including the interaction with pocket C, a region not commonly exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at the low nanomolar level in several tumoral settings, and to the selectivity toward IDO1 over TDO and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected by pancreatic ductal adenocarcinoma, was observed, pointing to this class of molecules as a valuable template for boosting the antitumor immune system.

The Cytokine Nicotinamide Phosphoribosyltransferase (eNAMPT; PBEF; Visfatin) Acts as a Natural Antagonist of C-C Chemokine Receptor Type 5 (CCR5).

(1) Background: Extracellular nicotinamide phosphoribosyltrasferase (eNAMPT) is released by various cell types with pro-tumoral and pro-inflammatory properties. In cancer, eNAMPT regulates tumor growth through the activation of intracellular pathways, suggesting that it acts through a putative receptor, although its nature is still elusive. It has been shown, using surface plasma resonance, that eNAMPT binds to the C-C chemokine receptor type 5 (CCR5), although the physiological meaning of this finding is unknown. The aim of the present work was to characterize the pharmacodynamics of eNAMPT on CCR5. (2) Methods: HeLa CCR5-overexpressing stable cell line and B16 melanoma cells were used. We focused on some phenotypic effects of CCR5 activation, such as calcium release and migration, to evaluate eNAMPT actions on this receptor. (3) Results: eNAMPT did not induce ERK activation or cytosolic Ca2+-rises alone. Furthermore, eNAMPT prevents CCR5 internalization mediated by Rantes. eNAMPT pretreatment inhibits CCR5-mediated PKC activation and Rantes-dependent calcium signaling. The effect of eNAMPT on CCR5 was specific, as the responses to ATP and carbachol were unaffected. This was strengthened by the observation that eNAMPT inhibited Rantes-induced Ca2+-rises and Rantes-induced migration in a melanoma cell line. (4) Conclusions: Our work shows that eNAMPT binds to CCR5 and acts as a natural antagonist of this receptor.

A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD+ salvage pathway in the nucleus.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.