Search results
Showing results for
Nicotinamide N-Methyl Transferase (NNMT) Sustains Innate Sensitivity to NAMPT Inhibition in YAP-dependent Stem-like/Mesenchymal Prostate Cancer.
Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway and a promising therapeutic target in cancer. Resistance to NAMPT inhibitors, such as FK866, remains a key limitation to their clinical translation. While acquired resistance in cancer cell lines has been linked to target mutations, increased drug efflux, and metabolic reprogramming, innate resistance mechanisms have been poorly studied. Addressing this gap is crucial for identifying patient subgroups that are most likely to benefit from NAMPT-targeted therapies. Advanced castration resistance prostate cancer (CRPC) lacks effective targeted treatments. Among its heterogeneous subtypes, stem cell-like CRPC (CRPC-SCL) is characterized by independence from androgen receptor (AR) signaling, dependency on YAP/TAZ, and mesenchymal traits. In this study, we identify the YAP/nicotinamide N-methyltransferase (NNMT) axis as a key regulator of innate sensitivity to FK866 in stem-like mesenchymal CRPC cells. Using genetic and pharmacological models, we show that YAP or NNMT silencing rescues PC3 cells from FK866-induced apoptosis, endoplasmic reticulum stress, and NAD(H) depletion. Metabolomic profiling confirmed that NNMT activity depletes nicotinamide, sensitizing cells to FK866. We further validated NNMT upregulation across clinical CRPC-SCL datasets, where it strongly correlates with mesenchymal and therapy-resistant phenotypes. Murine prostate cancer cells with mesenchymal/stemness phenotypes (DVL3-SCM), that exhibit NNMT overexpression and high aggressiveness in vivo, also show increased sensitivity to FK866 compared with their parental counterparts (DVL3-PAR). In conclusion, we identify the YAP/NNMT axis as a determinant of innate sensitivity to NAMPT inhibition in prostate cancer. These findings support the use of NNMT as a predictive biomarker for NAD+-targeting therapies and provide mechanistic insight into a metabolic vulnerability of the CRPC-SCL subtype. Targeting the YAP/NNMT/NAMPT axis may represent a novel strategy for treating stem-like/mesenchymal, therapy-resistant prostate cancers.
Design, synthesis and characterization of aryl bis-guanyl hydrazones as RNA binders of C9orf72 G4C2 extended repeats.
Expanded G4C2 repeats derived from mutations of the C9orf72 gene are causative factors in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, leading to multiple pathological events. Bis thiophene para dinicotinimidamide 2a was reported to preferentially stabilize G-quadruplex G4C2 RNA structures at sub-micromolar concentrations. We replaced its amidine groups with BBB-compliant guanyl hydrazones, and carried out scaffold variations to improve water solubility. An eight-membered array was built around bis-thiophene- (4b-6a), bis-oxazole- (7b), diphenylurea diamide- (8b) and phenyldioxy ditriazolephenyl scaffolds (9a,b). Biological profiling of the array identified 4b as a promising, drug-like hit, active in cellular assays on ALS patient-derived cells.
NAMPT and NNMT released via extracellular vesicles and as soluble mediators are distinguished traits of BRAF inhibitor resistance of melanoma cells impacting on the tumor microenvironment.
UNLABELLED: Drugs targeting mutant BRAF and MEK oncogenes are effective in melanoma, even though resistance rapidly develops. This complex picture includes acquired intrinsic tumor and tumor microenvironmental-mediated mechanisms. Here we show that melanoma cells resistant to BRAF inhibitors (BRAFi) overexpress the rate-limiting enzymes involved in nicotinamide (NAM) metabolism nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide N-methyltransferase (NNMT). Remarkably, these cells release NAMPT and NNMT both in the free-form or loaded into extracellular vesicles (EVs). NAMPT is emerging as a key mediator of resistance to BRAFi in melanoma, primarily due to its established role in NAD biosynthesis. Although previously identified as a soluble extracellular factor in this tumor, its presence within EVs released by melanoma cells has not been reported until now, highlighting a previously unrecognized mechanism through which NAMPT may influence the tumor microenvironment (TME). NNMT was revealed to increase in melanoma lesions compared to benign nevi. Here, we report for the first time its overexpression in resistant melanoma cell lines at intracellular and extracellular levels (secreted both as a soluble factor and into EVs). NNMT expression is increased in BRAF-mutated melanoma patients, suggesting a link between its upregulation and the BRAF oncogenic signaling. Moreover, NNMT levels positively correlate with gene signatures associated with pro-inflammatory signaling, immune cell migration, and chemokine-mediated pathways. NNMT pharmacological inhibition and genetic silencing significantly reduce resistant melanoma cell growth. In addition, we found that BRAFi-resistant cells are more sensitive to NNMT inhibition, highlighting a trait of vulnerability of BRAFi-resistant melanomas. Lastly, we proposed for the first time a tetrameric NNMT:TLR4 binding model offering a plausible structural and mechanistic basis for their association. Our functional results indicated that exogenous NNMT treatment is able to trigger NF-κB pathway, one of the main TLR4-dependent signaling, sharing this cytokine-like properties with NAMPT, and opening a future deeper exploration of its functional role in the extracellular space. Overall, the identification of NAMPT and, surprisingly also NNMT, included in EVs and abundantly released from resistant melanoma cells supports the impact of these moonlighting proteins involved in nicotinamide metabolism as mediators of BRAF/MEK inhibitors resistance with tumor intrinsic and potentially tumor microenvironment-mediated mechanisms. Interfering with nicotinamide metabolism could be a valid strategy to counteract drug resistance acting on the multifactorial tumor-host interactions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-025-02361-2.
Autophagy induction by piplartine ameliorates axonal degeneration caused by mutant HSPB1 and HSPB8 in Charcot-Marie-Tooth type 2 neuropathies.
HSPB1 [heat shock protein family B (small) member 1] and HSPB8 are essential molecular chaperones for neuronal proteostasis, as they prevent protein aggregation. Mutant HSPB1 and HSPB8 primarily harm peripheral neurons, resulting in axonal Charcot-Marie-Tooth neuropathies (CMT2). Macroautophagy/autophagy is a shared mechanism by which HSPB1 and HSPB8 mutations cause neuronal dysfunction. Autophagosome formation is reduced in mutant HSPB1-induced pluripotent stem-cell-derived motor neurons from CMT type 2F patients. Likewise, the HSPB8K141N knockin mouse model, mimicking CMT type 2 L, exhibits axonal degeneration and muscle atrophy, with SQSTM1/p62-positive deposits. We show here that mouse embryonic fibroblasts isolated from a HSPB8K141N/green fluorescent protein (GFP)-LC3 model have diminished autophagosome production under conditions of MTOR inhibition. To correct the autophagic deficits in the HSPB1 and HSPB8 models, we screened by high-throughput autophagosome quantification the repurposing Spectrum Collection library for molecules that could boost the autophagic activity above the canonical MTOR inhibition. Hit compounds were validated on motor neurons obtained by differentiation of HSPB1P182L and HSPB8K141N patient-derived induced pluripotent stem cells, focusing on autophagy induction as well as neurite network density, axonal degeneration, and mitochondrial morphology. We identified molecules that specifically stimulate autophagosome formation in the HSPB8K141N cells, without affecting autophagy flux. Two top lead compounds induced autophagy and reduced axonal degeneration, thus promoting neuronal network maturation in the CMT2 patient-derived motor neurons. Based on these findings, the phenotypical screen revealed that piplartine rescued autophagy deficiencies in both the HSPB1 and HSPB8 models, demonstrating autophagy induction as an effective therapeutic strategy for CMT neuropathies and other chaperonopathies.
Synthesis and Preliminary Evaluation of Tanshinone Mimic Conjugates for Mechanism of Action Studies.
Human antigen R (HuR) is an RNA binding protein (RBP) belonging to the ELAV (Embryonic Lethal Abnormal Vision) family, which stabilizes mRNAs and regulates the expression of multiple genes. Its altered expression or localization is related to pathological features such as cancer or inflammation. Dihydrotanshinone I (DHTS I) is a naturally occurring, tetracyclic ortho-quinone inhibitor of the HuR-mRNA interaction. Our earlier efforts led to the identification of a synthetic Tanshinone Mimic (TM) 2 with improved affinity for HuR. Here we report five new TM probes 3-5 bearing a detection-promoting moiety (either photo affinity probe - PAP or biotin) as a para-substituent on the phenyl-sulphonamide for mechanism of action (MoA) studies. Biological and biochemical assays were used to characterize the novel TM conjugates 3-5. They showed similar toxic activity in HuR-expressing triple-negative breast cancer MDA-MB-231 cells, with micromolar CC50s. REMSAs revealed that photoactivatable groups (4 a and 4 b), but not biotin (5 a and 5 b), prevented conjugates' ability to disrupt rHuR-RNA complexes. Further biochemical studies confirmed that biotinylated probes, in particular 5 a, can be used to isolate rM1 M2 from solutions, taking advantage of streptavidin-coated magnetic beads, thus being the most promising HuR inhibitor to be used for further MoA studies in cell lysates.
Characterization of a novel thermostable NAD+-dependent formate dehydrogenase from Methylacidiphilum kamchatkense Kam1 (MkaFDH)
Metal-independent NAD+-dependent formate dehydrogenases (FDHs) are enzymes responsible for catalyzing the conversion of formate (HCOO–) to carbon dioxide (CO2), a biological reaction involved in microbial carbon processing and cofactor regeneration. These enzymes show large potential for environmental bioremediation and biotechnological uses. However, FDHs applications are hampered by the enzymes’ limited stability under extreme conditions, such as high temperatures or extreme pH. Therefore, we aimed to identify and characterize novel metal-independent FDHs with improved activity and thermostability compared to known FDHs. By using four different FDH protein sequences, CtFDH (from Chaetomium thermophilum), MtFDH (from Myceliophthora thermophile), OpFDH (from Ogata parapolymorpha DL-1) and PseFDH (from Pseudomonas sp.101) we retrieved 18,850 FDHs sequences from the NCBI database and matched against the species present in the database of thermophilic bacteria, ThermoBase. Our phylogenetic analysis identified four distinct FDHs in thermophilic bacteria: Methylocaldum szegediense (MszFDH), Methylacidiphilum kamchatkense (MkaFDH), Mycobacterium arosiense (MarFDH) and Mycobacterium genavense (MgeFDH). We selected and characterized the MkaFDH as it was expressed in the thermophilic bacterium with the highest optimum growth (55 °C) among the four bacteria. The MkaFDH was cloned, and the recombinant protein was expressed in E. coli and purified. The conditions for the optimal catalytic activity for formate oxidations were screened and identified, revealing metal-independent, NAD+-restricted activity in phosphate buffer, pH 8. Importantly, the enzyme showed remarkable thermal stability and catalytic activity, showing a melting temperature (Tm) of 60.15 °C, as confirmed by far-UV circular dichroism (CD). Finally, the enzyme showed good thermostability for formate oxidation up to 57.5 °C, and its high catalytic efficiency (kcat/Km = 0.44 s−1mM−1) suggested its potential industrial application. Collectively, we describe here a novel FDH with relevant thermostability that can be exploited as a prototype for industrial applications.
Novel, soluble 3-heteroaryl-substituted tanshinone mimics attenuate the inflammatory response in murine macrophages.
The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR - 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b.
CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells.
BACKGROUND: Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications. METHODS: In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors. RESULTS: Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies. CONCLUSIONS: This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.
Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.
Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.