Discovery of a novel NAMPT inhibitor that selectively targets NAPRT-deficient EMT-subtype cancer cells and alleviates chemotherapy-induced peripheral neuropathy.

Background: Exploiting synthetic lethality (SL) relationships between protein pairs has emerged as an important avenue for the development of anti-cancer drugs. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of the NAD+ salvage pathway, having an SL relationship with nicotinic acid phosphoribosyltransferase (NAPRT), the key enzyme in the NAD+ Preiss-Handler pathway. NAMPT inhibitor holds clinical potential not only as a promising cancer treatment but also as a means of protection against chemotherapy-induced-peripheral-neuropathy (CIPN). However, as NAD+ is essential for normal cells, the clinical use of NAMPT inhibitors is challenging. This study aimed to identify a novel NAMPT inhibitor with enhanced selective cytotoxicity against NAPRT-deficient cancer cells as well as prominent efficacy in alleviating CIPN. Methods: We began by conducting drug derivatives screening in a panel of lung cancer cell lines to select an agent with the broadest therapeutic window between the NAPRT-negative and-positive cancer cell lines. Both in vitro and In vivo comparative analyses were conducted between A4276 and other NAMPT inhibitors to evaluate the NAPRT-negative cancer cell selectivity and the underlying distinct NAMPT inhibition mechanism of A4276. Patient-derived tumor transcriptomic data and protein levels in various cancer cell lines were analyzed to confirm the correlation between NAPRT depletion and epithelial-to-mesenchymal transition (EMT)-like features in various cancer types. Finally, the efficacy of A4276 for axonal protection and CIPN remedy was examined in vitro and in vivo. Results: The biomarker-driven phenotypic screening led to a discovery of A4276 with prominent selectivity against NAPRT-negative cancer cells compared with NAPRT-positive cancer cells and normal cells. The cytotoxic effect of A4276 on NAPRT-negative cells is achieved through its direct binding to NAMPT, inhibiting its enzymatic function at an optimal and balanced level allowing NAPRT-positive cells to survive through NAPRT-dependent NAD+ synthesis. NAPRT deficiency serves as a biomarker for the response to A4276 as well as an indicator of EMT-subtype cancer in various tumor types. Notably, A4276 protects axons from Wallerian degeneration more effectively than other NAMPT inhibitors by decreasing NMN-to-NAD+ ratio. Conclusion: This study demonstrates that A4276 selectively targets NAPRT-deficient EMT-subtype cancer cells and prevents chemotherapy-induced peripheral neuropathy, highlighting its potential as a promising anti-cancer agent for use in cancer monotherapy or combination therapy with conventional chemotherapeutics.

mRNA transport, translation, and decay in adult mammalian central nervous system axons.

Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knockout mice validate FMRP's role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the adult axonal transcriptome is stably maintained by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function in vivo.

Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo.

Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated.

GABBR2 mutations determine phenotype in rett syndrome and epileptic encephalopathy.

OBJECTIVE: Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. METHODS: We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole-exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. RESULTS: We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. INTERPRETATION: GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated γ-aminobutyric acid signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. Ann Neurol 2017;82:466-478.

Dynamic Axonal Translation in Developing and Mature Visual Circuits.

Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.

Disruption of Pten speeds onset and increases severity of spontaneous colitis in Il10(-/-) mice.

BACKGROUND & AIMS: Early-onset ulcerative colitis, which is considered severe colonic inflammation that develops in infants and young children, can be caused by alterations in interleukin (IL)-10 signaling, although other factors are involved in its pathogenesis. We investigated whether loss of phosphatase and tensin homologue (PTEN), which regulates many important cell functions such as cell proliferation, cell survival, and Toll-like receptor (TLR) signaling pathways, contributes to the development of colitis in Il10(-/-) mice. METHODS: We generated Il10(-/-) mice (in C57BL/6 and C3H/HeJBir background strains) with disruption of Pten in the intestinal epithelium (Ints(ΔPten/ΔPten);Il10(-/-) mice) and Ints(ΔCont);Il10(-/-) (control) mice. Colon tissues were collected and histological, transmission electron microscopy, and gene expression analysis were performed. Fecal microbiota samples were analyzed by sequencing of 16S ribosomal RNA genes. We disrupted Tlr4 in Ints(ΔPten/ΔPten);Il10(-/-) mice. Lipopolysaccharide signaling via TLR4 was blocked by treating mice with polymyxin B. RESULTS: Il10(-/-) mice developed colitis when they were 6 to 7 months old, whereas Ints(ΔPten/ΔPten);Il10(-/-) mice developed severe colitis and colon tumors by the time they were 36 days old. Within 3 months of birth, 80% of Ints(ΔPten/ΔPten);Il10(-/-) mice developed severe colitis and colonic malignancy, whereas none of the Ints(ΔCont);Il10(-/-) mice had these phenotypes. Ints(ΔPten/ΔPten);Il10(-/-) mice had alterations in fecal microbiota compared with controls, such as increased proportions of Bacteroides species, which are gram negative. Disruption of Tlr4 or treating Ints(ΔPten/ΔPten);Il10(-/-) mice with polymyxin B delayed the development of colitis and reduced disease severity. CONCLUSIONS: Disruption of Pten in the intestinal epithelium of Il10(-/-) mice speeds the onset and increases the severity of colitis. Fecal microbiota from Ints(ΔPten/ΔPten);Il10(-/-) mice have increased proportions of Bacteroides species. Development of colitis is delayed and reduced by blocking TLR4 signaling. Ints(ΔPten/ΔPten);Il10(-/-) mice may be studied as a model for early-onset ulcerative colitis and used to identify new therapeutic targets.

PTEN regulates TLR5-induced intestinal inflammation by controlling Mal/TIRAP recruitment.

Defective IL-10 allele is a risk factor for intestinal inflammation. Indeed, IL-10(-/-) mice are predisposed to spontaneous colitis in the presence of intestinal microbiota, indicating that microbial factors contribute to developing intestinal inflammation. By recognizing flagellin, TLR5 plays a quintessential role in microbial recognition in intestinal epithelial cells. Here, we treated flagellin (1.0 µg/mouse/d) in mouse colon and found that it elicited colonic inflammation in IL-10(-/-) mice, characterized with tissue hypertrophy, inflamed epithelium, and enhanced cytokine production in the colon (MPO, KC, IL-6; ≥2-fold; P < 0.05). These inflammatory effects were dramatically inhibited in TLR5(-/-);IL-10(-/-) mice. Intestinal epithelium specific PTEN deletion significantly attenuated flagellin-promoted colonic inflammation in IL-10(-/-) mice. As a molecular mechanism that PTEN deletion inhibited TLR5-elicited responses, we hypothesized that PTEN regulated TLR5-induced responses by controlling the involvement of Mal in TLR5 engagement. Mal interacted with TLR5 on flagellin, and Mal deficiency inhibited flagellin-induced responses in intestinal epithelial cells. Similarly, Mal(-/-);IL-10(-/-) mice showed reduced flagellin-promoted responses. Furthermore, PTEN deletion disrupted Mal-TLR5 interaction, resulting in diminished TLR5-induced responses. PTEN deletion impeded Mal localization at the plasma membrane and suppressed Mal-TLR5 interaction. These results suggest that, by controlling Mal recruitment, PTEN regulates TLR5-induced inflammatory responses.

Toll-like receptor 5 engagement induces interleukin-17C expression in intestinal epithelial cells.

The family of interleukin-17 (IL-17) cytokine is the essential inflammatory mediator that influences the pathophysiology of various inflammatory diseases. Many studies focused on investigating the expression, signaling, and biological impacts of IL-17A and IL-17F, and the neutralization of these cytokines exhibited some promising results in clinical trials. In contrast, the expression resources and physiological relevance of IL-17C remained to be studied. In this study, through a microarray approach conducted with nontransformed human colonic epithelial cells (NCM460), we found that bacterial flagellin stimulation elicited potent IL-17C mRNA expression. We also confirmed that IL-17C protein production was strongly induced by flagellin in these cells. Flagellin-induced IL-17C expression was also observed in human colon adenocarcinoma cells such as DLD-1 and HT-29, indicating that IL-17C could be a signature inflammatory cytokine from intestinal epithelial cells in response to flagellin. Since inhibited in TLR5-, or MyD88- or TRIF-silenced cells, flagellin-induced IL-17C expression was specifically mediated by TLR5 and, subsequently, MyD88 and TRIF adaptor molecules. Furthermore, in line with inflammatory nature of IL-17, we found that IL-17C expression was substantially enhanced in the intestinal tissues from Ulcerative colitis patients. Given the facts that TLR5 is a key pattern recognition receptor which mediates microbial recognition in the intestinal epithelium and IL-17C turned out to be a unique member of the IL-17 family expressed in intestinal epithelial cells on TLR5 activation, our study may provide an important clue on understanding how intestinal microbes would contribute to an inflammatory program in the gut.

The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase.

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC-RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.

Regulation of Notch1 signaling by Delta-like ligand 1 intracellular domain through physical interaction.

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. The Delta-like ligand 1 also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. In this study, we have demonstrated that the Delta-like 1 intracellular domain (Dll1-IC) specifically binds to Notch1-IC in the nucleus, thereby disrupting the association of the Notch1-IC-RBP-Jk-MAM transcription activator complex. Additionally, the Notch1-mediated blockage of the induction of MyoD is abolished by the co-expression of Dll1-IC. Collectively, our results show that Dll1-IC functions as a negative regulator in Notch signaling via the disruption of the Notch1-IC-RBP-Jk complex.

Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.

The Notch1 receptor is a crucial controller of cell fate decisions, and is also a key regulator of cell growth and differentiation in a variety of contexts. In this study, we have demonstrated that the APP intracellular domain (AICD) attenuates Notch1 signaling by accelerated degradation of the Notch1 intracellular domain (Notch1-IC) and RBP-Jk, through different degradation pathways. AICD suppresses Notch1 transcriptional activity by the dissociation of the Notch1-IC-RBP-Jk complex after processing by γ-secretase. Notch1-IC is capable of forming a trimeric complex with Fbw7 and AICD, and AICD enhances the protein degradation of Notch1-IC through an Fbw7-dependent proteasomal pathway. AICD downregulates the levels of RBP-Jk protein through the lysosomal pathway. AICD-mediated degradation is involved in the preferential degradation of non-phosphorylated RBP-Jk. Collectively, our results demonstrate that AICD functions as a negative regulator in Notch1 signaling through the promotion of Notch1-IC and RBP-Jk protein degradation.

Inhibition of Notch1 signaling by Runx2 during osteoblast differentiation.

Notch1 genes encode receptors for a signaling pathway that regulates cell growth and differentiation in various contexts, but the role of Notch1 signaling in osteogenesis is not well defined. Notch1 controls osteoblast differentiation by affecting Runx2, but the question arises whether normal osteoblastic differentiation can occur regardless of the presence of Notch1. In this study, we observed the downregulation of Notch1 signaling during osteoblastic differentiation. BMPR-IB/Alk6-induced Runx2 proteins reduced Notch1 activity to a marked degree. Accumulated Runx2 suppressed Notch1 transcriptional activity by dissociating the Notch1-IC-RBP-Jk complex. Using deletion mutants, we also determined that the N-terminal domain of Runx2 was crucial to the binding and inhibition of the N-terminus of the Notch1 intracellular domain. Notably, upregulation of the Runx2 protein level paralleled reduced expression of Hes1, which is a downstream target of Notch1, during osteoblast differentiation. Collectively, our data suggest that Runx2 is an inhibitor of the Notch1 signaling pathway during normal osteoblast differentiation.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) controls Notch1 signaling by downregulation of protein stability through Fbw7 ubiquitin ligase.

Notch is a transmembrane protein that acts as a transcriptional factor in the Notch signaling pathway for cell survival, cell death and cell differentiation. Notch1 and Fbw7 mutations both lead the activation of the Notch1 pathway and are found in the majority of patients with the leukemia T-ALL. However, little is known about the mechanisms and regulators that are responsible for attenuating the Notch signaling pathway through Fbw7. Here, we report that the serum- and glucocorticoid-inducible protein kinase SGK1 remarkably reduced the protein stability of the active form of Notch1 through Fbw7. The protein level and transcriptional activity of the Notch1 intracellular domain (Notch1-IC) were higher in SGK1-deficient cells than in SGK1 wild-type cells. Notch1-IC was able to form a trimeric complex with Fbw7 and SGK1, thereby SGK1 enhanced the protein degradation of Notch1-IC via a Fbw7-dependent proteasomal pathway. Furthermore, activated SGK1 phosphorylated Fbw7 at serine 227, an effect inducing Notch1-IC protein degradation and ubiquitylation. Moreover, accumulated dexamethasone-induced SGK1 facilitated the degradation of Notch1-IC through phosphorylation of Fbw7. Together our results suggest that SGK1 inhibits the Notch1 signaling pathway via phosphorylation of Fbw7.

DJ-1 modulates the p38 mitogen-activated protein kinase pathway through physical interaction with apoptosis signal-regulating kinase 1.

DJ-1 has been reported as a gene linked to early onset familial Parkinson's disease, and is functionally involved in transcriptional regulation and oxidative stress-induced cell death. To understand the role of DJ-1 in cellular stress, this study investigated DJ-1's effect on stress-activated protein kinase signaling and H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). According to the results, the overexpression of DJ-1 inhibited H(2)O(2)-induced activation of ASK1 as well as the activation of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. The results of both in vivo binding and kinase studies have revealed that ASK1 is the direct target of DJ-1, whereas it has shown no effect on either MKK3 or p38. DJ-1 blocked both the homo-oligomerization of ASK1 and inhibited ASK1 activity. Taken together, our data strongly suggest that DJ-1, by directly inhibiting ASK1, may act as a negative regulator in ASK1 signaling cascades.