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Neural stem/progenitor cells (NSPCs) contribute to the physiological cellular turnover of the adult brain and make up its regenerative potential. It is thus essential to understand how different factors influence their proliferation and differentiation to gain better insight into potential therapeutic targets in neurodegenerative diseases and traumatic brain injuries. Recent evidences indicate the roles of redox stress sensing and coping mechanisms in mediating the balance between NSPC self-renewal and differentiation. Such mechanisms involve direct cysteine modification, signaling and metabolic reprogramming, epigenetic alterations and transcription changes leading to adaptive responses like autophagy. Here, we discuss emerging findings on the involvement of redox sensors and effectors and their mechanisms in influencing changes in cellular redox potential and NSPC fate.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFβ/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFβ secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFβ-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. Significance: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.

Despite recent therapeutic advances, prostate cancer remains a leading cause of cancer-related death. A subset of castration-resistant prostate cancers become androgen receptor (AR) signaling independent and develop neuroendocrine prostate cancer (NEPC) features through lineage plasticity. These NEPC tumors, associated with aggressive disease and poor prognosis, are driven, in part, by aberrant expression of N-Myc, through mechanisms that remain unclear. Integrative analysis of the N-Myc transcriptome, cistrome, and interactome using in vivo, in vitro, and ex vivo models (including patient-derived organoids) identified a lineage switch towards a neural identity associated with epigenetic reprogramming. N-Myc and known AR cofactors (e.g., FOXA1 and HOXB13) overlapped, independently of AR, at genomic loci implicated in neural lineage specification. Moreover, histone marks specifically associated with lineage-defining genes were reprogrammed by N-Myc. We also demonstrated that the N-Myc–induced molecular program accurately classifies our cohort of patients with advanced prostate cancer. Finally, we revealed the potential for enhancer of zeste homolog 2 (EZH2) inhibition to reverse the N-Myc–induced suppression of epithelial lineage genes. Altogether, our data provide insights into how N-Myc regulates lineage plasticity and epigenetic reprogramming associated with lineage specification. The N-Myc signature we defined could also help predict the evolution of prostate cancer and thus better guide the choice of future therapeutic strategies.

Epidermal growth factor receptor (EGFR) is frequently amplified, mutated and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical response, and the underlying mechanism remains poorly understood. Through analyses of an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals a small population of glioma cells that can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses further identify TGFβ/YAP/Slug signaling cascade activation as major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, the TGFβ secreted from the stressed glioma cells acts to promote YAP nuclear translocation and activation, which subsequently stimulates upregulation of the pro-mesenchymal transcriptional factor Slug and then glioma lineage transdifferentiation towards a stable therapy-refractory state. Blockade of this adaptive response through enforced dominant negative YAP expression significantly delayed anti-EGFR relapse and significantly prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses.SignificanceThis study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and is clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas carrying aberrant EGFR signaling.

Capicua (CIC), a member of the high mobility group-box (HMG-box) superfamily of transcriptional repressors, is frequently mutated in human oligodendrogliomas. However, its functions in brain development and tumorigenesis remain poorly understood. Here, we report that brain-specific deletion of Cic compromises developmental transition of neuroblasts to immature neurons in mouse hippocampus and compromises normal neuronal differentiation. Combined gene expression and ChIP-seq analyses identified VGF as an important CIC-repressed transcriptional surrogate involved in neuronal lineage regulation. Aberrant VGF expression promotes neural progenitor cell proliferation by suppressing their differentiation. Mechanistically, we demonstrated that CIC represses VGF expression by tethering SIN3-HDAC to form a transcriptional corepressor complex. Mass spectrometry analysis of CIC-interacting proteins further identified the BRG1-containing mSWI/SNF complex whose function is necessary for transcriptional repression by CIC. Together, this study uncovers a potentially novel regulatory pathway of CIC-dependent neuronal differentiation and may implicate these molecular mechanisms in CIC-dependent brain tumorigenesis.

Summary Loss of a cell's ability to terminally differentiate because of mutations is a selected genetic event in tumorigenesis. Genomic analyses of low-grade glioma have reported recurrent mutations of far upstream element-binding protein 1 (FUBP1). Here, we show that FUBP1 expression is dynamically regulated during neurogenesis and that its downregulation in neural progenitors impairs terminal differentiation and promotes tumorigenesis collaboratively with expression of IDH1R132H. Mechanistically, collaborative action between SRRM4 and FUBP1 is necessary for mini-exon splicing of the neurospecific LSD1+8a isoform. LSD1+8a was downregulated upon loss of FUBP1 in neural progenitors, thereby impairing terminal neuronal differentiation and maturation. Reinforcing LSD1+8a expression in FUBP1-downregulated neural progenitors restored terminal differentiation and suppressed tumorigenesis; hence, LSD1+8a is an obligatory effector of FUBP1-dependent neuronal differentiation. These findings establish a direct role for FUBP1 in neuronal differentiation and also explain its tumor-suppressor function in the nervous system., Graphical Abstract, Highlights • FUBP1 is the tumor suppressor necessary for terminal differentiation of neurons • FUBP1 promotes inclusion of neuron-specific LSD1 exon 8a • FUBP1 and IDH1 mutations converge on impaired differentiation of neural progenitors, In this article, Paik and colleagues demonstrate that FUBP1 plays an indispensable role in promoting terminal differentiation of neurons and that lack of FUBP1 interferes with early-born neuronal cells exiting the cell cycle and predisposes these cells for transformation. These findings explain how FUBP1 serves uniquely as a tumor suppressor in the CNS.

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, comprising 5% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. The average overall survival of patients with MCL is 4-6 years and for the majority of patients who progress on targeted agents, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated to improve treatment outcomes and quality of life. The type II protein arginine methyltransferase enzyme, PRMT5 is overexpressed and promotes growth and survival of MCL. Inhibition of PRMT5 with a novel, SAM-competitive class of inhibitors drives anti-tumor activity in MCL cell lines and patient derived xenograft (PDX) models derived from patients with relapse or refractory disease. Selective inhibition of PRMT5 with PRT-382 (Prelude Therapeutics) in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to target genes. By performing chromatin immunoprecipitation-sequencing (ChIP seq) analysis, we identified over 800 newly emerged FOXO1-bound genomic loci, including multiple pro-apoptotic BCL2 family proteins (BAX, BAK1, BIK, BBC3, BMF and NOXA1). FOXO1 localization and transcriptional differences were confirmed by ChIP PCR and RT-PCR respectively. Protein levels were measured with Western blotting. BAX was identified as the most common direct target of FOXO1-transriptional activity that was upregulated on both a transcript and protein level. This led us to hypothesize that PRMT5 inhibition could potentially drive a therapeutic vulnerability to the BCL-2 inhibitor venetoclax. Single agent and combination treatment with venetoclax and PRT382 was performed in nine MCL lines. Of the nine lines, four were considered relatively resistant to PRT-382 and five resistant to venetoclax. Synergy scores, determined from MTS assays, showed significant levels of synergy in the majority of MCL lines tested. CCMCL1 and UPN1, BCL-2 negative MCL lines, and Maver1, which is highly resistant to PRMT5i, were the only cell lines to not show synergy. The cell line with the highest levels of synergy, Z-138, expressed high levels of BCL-2 and is ibrutinib resistant. Overall, there was a strong positive correlation between BCL-2 expression and synergy score (r= -0.8956, p=0.0064). The synergy seen was confirmed to be through the intrinsic apoptotic pathway based on caspase activity. To determine a mechanism of action, BAX and BAK1 were knocked down in four cell lines, three that displayed synergy and one that was resistant. This suggests that BAX expression is essential for synergy between PRMT5 and BCL2 inhibition to occur. Knock down of BAK1, the other effector of the BCL2 family of proteins, did not show protection suggesting that BAX is necessary and sufficient for this therapeutic synergy to occur. We also determined that p53 status did not correlate to the response seen (p=0.477), supporting that this mechanism is occurring through FOXO1 transcriptional regulation. In vivo evaluation in two preclinical MCL models showed therapeutic synergy with combination venetoclax/PRT382 treatment. Mice were treated with sub-therapeutic doses of venetoclax and/or PRT382 and disease burden was assessed weekly via flow cytometry. Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in the MCL in vivo models showed synergistic anti-tumor activity. Both PDX models showed an extension of life with combination treatment (P Disclosures Zhang: Prelude Therapeutics: Current Employment. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: AstraZeneca: Research Funding; Janssen: Research Funding; Johnson and Johnson: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Eli Lilly: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Paik: Forkhead BioTherapeutics: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Capicua (CIC), a member of the high mobility group (HMG)-box superfamily of transcriptional repressors, is frequently mutated in human oligodendrogliomas. But its function in brain development and tumorigenesis remains poorly understood. Here, we report that brain-specific deletion of Cic compromises developmental transition of neuroblast to immature neurons in mouse hippocampus and compromises normal neuronal differentiation. Combined gene expression and ChIP-seq analyses identified VGF as an important CIC-repressed transcriptional surrogates involved in neuronal lineage regulation. Aberrant VGF expression promotes neural progenitor cell proliferation by suppressing their differentiation. Mechanistically, we demonstrated that CIC represses VGF expression by tethering SIN3-HDAC to form a transcriptional corepressor complex. Mass spectrometry analysis of CIC-interacting proteins further identified BRG1 containing mSWI/SNF complex of which function is necessary for transcriptional repression by CIC. Together, this study uncovers a novel regulatory pathway of CIC-dependent neuronal differentiation and provides molecular insights into the etiology of CIC-dependent brain tumors.

Peroxisomes are essential organelles for maintaining the homeostasis of lipids and reactive oxygen species (ROS). While oxidative stress-induced endoplasmic reticulum (ER) stress plays an important role in nonalcoholic fatty liver disease (NAFLD), the role of peroxisomes in ROS-mediated ER stress in the development of NAFLD remains elusive. We investigated whether an impaired peroxisomal redox state accelerates NAFLD by activating ER stress by inhibiting catalase, an antioxidant expressed exclusively in peroxisomes. Wild-type (WT) and catalase knockout (CKO) mice were fed either a normal diet or a high-fat diet (HFD) for 11 weeks. HFD-induced phenotype changes and liver injury accompanied by ER stress and peroxisomal dysfunction were accelerated in CKO mice compared to WT mice. Interestingly, these changes were also significantly increased in CKO mice fed a normal diet. Inhibition of catalase by 3-aminotriazole in hepatocytes resulted in the following effects: (i) increased peroxisomal H2O2 levels as measured by a peroxisome-targeted H2O2 probe (HyPer-P); (ii) elevated intracellular ROS; (iii) decreased peroxisomal biogenesis; (iv) activated ER stress; (v) induced lipogenic genes and neutral lipid accumulation; and (vi) suppressed insulin signaling cascade associated with JNK activation. N-acetylcysteine or 4-phenylbutyric acid effectively prevented those alterations. These results suggest that a redox imbalance in peroxisomes perturbs cellular metabolism through the activation of ER stress in the liver.

Summary Neurodegeneration resulting in cognitive and motor impairment is an inevitable consequence of aging. Little is known about the genetic regulation of this process despite its overriding importance in normal aging. Here, we identify the Forkhead Box O (FOXO) transcription factor 1, 3, and 4 isoforms as a guardian of neuronal integrity by inhibiting age‐progressive axonal degeneration in mammals. FOXO expression progressively increased in aging human and mouse brains. The nervous system‐specific deletion of Foxo transcription factors in mice accelerates aging‐related axonal tract degeneration, which is followed by motor dysfunction. This accelerated neurodegeneration is accompanied by levels of white matter astrogliosis and microgliosis in middle‐aged Foxo knockout mice that are typically only observed in very old wild‐type mice and other aged mammals, including humans. Mechanistically, axonal degeneration in nerve‐specific Foxo knockout mice is associated with elevated mTORC1 activity and accompanying proteotoxic stress due to decreased Sestrin3 expression. Inhibition of mTORC1 by rapamycin treatment mimics FOXO action and prevented axonal degeneration in Foxo knockout mice with accelerated nervous system aging. Defining this central role for FOXO in neuroprotection during mammalian aging offers an invaluable window into the aging process itself.

Loss of terminal differentiation capacity through genetic mutations is a selected genetic event in tumorigenesis. 1p19q co-deletion associated recurrent mutations of far upstream element-binding protein 1 (FUBP1) have been reported from genomic analysis of low- grade oligodendroglioma. It is frequently overexpressed in many human cancers and strongly correlated with disease progression. However, left unanswered is how FUBP1 serves uniquely as a tumor suppressor in the central nervous system. Here we show that expression of FUBP1 is dynamically regulated during neurogenesis, marking specific stages of neuronal differentiation. Loss of FUBP1 in neural stem/progenitor cells (NPC) increased the number of cells fail to terminally differentiate. Knockdown of FUBP1 further blocks differentiation and promotes tumorigenesis collaboratively with the expression of neomorphic isocitrate dehydrorgenase 1 mutant IDH1R132H. Mechanistically FUBP1 is necessary for mini exon 8a splicing of neurospecific isoform of histone demethylase LSD1 (nLSD1). It binds to intron 8 of LSD1 together with a neuron-specific splicing complex containing NOVA1 and 2 and works synergistically with SRRM4 for the inclusion of mini exon 8a. The expression level of nLSD1 is decreased upon loss of FUBP1 in neural progenitors, impairing the terminal neuronal differentiation and maturation. Reinforcing nLSD1 expression in FUBP1-downregulated NPC restores terminal differentiation, establishing nLSD1 as an obligatory effector of FUBP1-dependent neuronal differentiation. Together, these findings establish a direct role for FUBP1 in neuronal differentiation and also uncover the mechanism for its tumor suppressor function in the nervous system.

Forkhead transcription factor family O (FoxO) maintains adult stem cell reserves by supporting their long-term proliferative potential. MicroRNAs (miRs) regulate neuronal stem/progenitor cell (NSPC) proliferation and differentiation during neural development by controlling the expression of a specific set of target genes. In the neurogenic subventricular zone, FoxO1 is specifically expressed in NSPCs and is no longer detected during the transition to neuroblast stage, forming an inverse correlation with miR-9 expression. The 3'-untranslated region of FoxO1 contains a conserved target sequence of miR-9 and FoxO1 expression is coordinated in concert with miR-9 during neuronal differentiation. Our study demonstrates that FoxO1 contributes to NSPC fate decision through its cooperation with the Notch signaling pathway.

Loss of the histone H3.3-specific chaperone component ATRX or its partner DAXX frequently occurs in human cancers that employ alternative lengthening of telomeres (ALT) for chromosomal end protection, yet the underlying mechanism remains unclear. Here, we report that ATRX/DAXX does not serve as an immediate repressive switch for ALT. Instead, ATRX or DAXX depletion gradually induces telomere DNA replication dysfunction that activates not only homology-directed DNA repair responses but also cell cycle checkpoint control. Mechanistically, we demonstrate that this process is contingent on ATRX/DAXX histone chaperone function, independently of telomere length. Combined ATAC-seq and telomere chromatin immunoprecipitation studies reveal that ATRX loss provokes progressive telomere decondensation that culminates in the inception of persistent telomere replication dysfunction. We further show that endogenous telomerase activity cannot overcome telomere dysfunction induced by ATRX loss, leaving telomere repair-based ALT as the only viable mechanism for telomere maintenance during immortalization. Together, these findings implicate ALT activation as an adaptive response to ATRX/DAXX loss-induced telomere replication dysfunction.

Truncated N(6)-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4'-thioadenosines. Hydrophobic N(6) and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N(6)-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f-4i did not differ significantly, with Ki values of 7.8-16.0 nM. N(6)-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

Macrophages are important components of adipose tissue inflammation, which results in metabolic diseases such as insulin resistance. Notably, obesity induces a proinflammatory phenotypic switch in adipose tissue macrophages, and oxidative stress facilitates this switch. Thus, we examined the role of endogenous catalase, a key regulator of oxidative stress, in the activity of adipose tissue macrophages in obese mice. Catalase knockout (CKO) exacerbated insulin resistance, amplified oxidative stress, and accelerated macrophage infiltration into epididymal white adipose tissue in mice on normal or high-fat diet. Interestingly, catalase deficiency also enhanced classical macrophage activation (M1) and inflammation but suppressed alternative activation (M2) regardless of diet. Similarly, pharmacological inhibition of catalase activity using 3-aminotriazole induced the same phenotypic switch and inflammatory response in RAW264.7 macrophages. Finally, the same phenotypic switch and inflammatory responses were observed in primary bone marrow-derived macrophages from CKO mice. Taken together, the data indicate that endogenous catalase regulates the polarization of adipose tissue macrophages and thereby inhibits inflammation and insulin resistance.

Mitochondrial reactive oxygen species (ROS) play an important role in diabetes complications, including diabetic nephropathy (DN). Plasma free fatty acids (FFAs) as well as glucose are increased in diabetes, and peroxisomes and mitochondria participate in FFA oxidation in an interconnected fashion. Therefore, we investigated whether deficiency of catalase, a major peroxisomal antioxidant, accelerates DN through peroxisomal dysfunction and abnormal renal FFA metabolism. Diabetes was induced by multiple injections of low-dose streptozotocin into catalase knock-out (CKO) and wild-type (WT) C57BL/6 mice. Murine mesangial cells (MMCs) transfected with catalase small interfering RNA followed by catalase overexpression were used to further elucidate the role of endogenous catalase. Despite equivalent hyperglycemia, parameters of DN, along with markers of oxidative stress, were more accelerated in diabetic CKO mice than in diabetic WT mice up to 10 weeks of diabetes. CKO mice and MMCs showed impaired peroxisomal/mitochondrial biogenesis and FFA oxidation. Catalase deficiency increased mitochondrial ROS and fibronectin expression in response to FFAs, which were effectively restored by catalase overexpression or N-acetylcysteine. These data provide unprecedented evidence that FFA-induced peroxisomal dysfunction exacerbates DN and that endogenous catalase plays an important role in protecting the kidney from diabetic stress through maintaining peroxisomal and mitochondrial fitness.

Loss of terminal differentiation capacity through genetic mutations is a selected genetic event in tumorigenesis. 1p19q co-deletion associated recurrent mutations of far upstream element-binding protein 1 (FUBP1) have been reported from genomic analysis of low- grade oligodendroglioma. It is frequently overexpressed in many human cancers and strongly correlated with disease progression. However, left unanswered is how FUBP1 serves uniquely as a tumor suppressor in the central nervous system. Here we show that expression of FUBP1 is dynamically regulated during neurogenesis, marking specific stages of neuronal differentiation. Loss of FUBP1 in neural stem/progenitor cells (NPC) increased the number of cells fail to terminally differentiate. Knockdown of FUBP1 further blocks differentiation and promotes tumorigenesis collaboratively with the expression of neomorphic isocitrate dehydrorgenase 1 mutant IDH1R132H. Mechanistically FUBP1 is necessary for mini exon 8a splicing of neurospecific isoform of histone demethylase LSD1 (nLSD1). It binds to intron 8 of LSD1 together with a neuron-specific splicing complex containing NOVA1 and 2 and works synergistically with SRRM4 for the inclusion of mini exon 8a. The expression level of nLSD1 is decreased upon loss of FUBP1 in neural progenitors, impairing the terminal neuronal differentiation and maturation. Reinforcing nLSD1 expression in FUBP1-downregulated NPC restores terminal differentiation, establishing nLSD1 as an obligatory effector of FUBP1-dependent neuronal differentiation. Together, these findings establish a direct role for FUBP1 in neuronal differentiation and also uncover the mechanism for its tumor suppressor function in the nervous system. Citation Format: Inah Hwang, Dongqing Cao, Do-Yeon Kim, Tuo Zhang, Jian Hu, Yu Yao, Jihye Paik. Loss of FUBP1 impairs terminal neuronal differentiation and predisposes neural progenitors for transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2481.

Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A 3 adenosine receptor (A 3 AR) is up-regulated under stress, but the therapeutic effects of A 3 AR antagonists on chronic kidney disease are not fully understood. The present study examined the effect of LJ-1888 [(2 R ,3 R ,4 S )-2-[2-chloro-6-(3-iodobenzylamino)-9 H -purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A 3 AR antagonist, on unilateral ureteral obstruction (UUO)–induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equivalent therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A 3 AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)–induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A 3 AR blockade. UUO- or TGF-β1–induced c-Jun N-terminal kinase and extracellular signal–regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A 3 AR blockade reduced UUO- or TGF-β1–induced up-regulation of lysyl oxidase, which induces cross-linking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A 3 AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.

Fibrosis of epithelial parenchymal organs and end-stage organ failure, the final common pathway of many progressive chronic diseases including chronic kidney disease, continue to increase worldwide and are a major determinant of morbidity and mortality. Fibrosis is an active biosynthetic healing response initiated to protect the tissue from injury through the timed release of proteins but leads to serious tissue damage when it becomes independent from the initiating stimulus. Massive deposition of extracellular matrix by accumulation of myofibroblasts and disruption of the normal tissue architecture are characteristic of tissue fibrosis. The highly conserved Wnt/beta-catenin signaling pathway is essential to embryonic development in general and kidney morphogenesis in particular by regulating the expression of target genes, most often through the transcription factor T cell factor (TCF) and/or lymphoid enhancer factor (LEF). Emerging evidence from studies of renal fibrosis suggests that altered Wnt/beta-catenin signaling is linked to the pathogenesis of renal fibrosis. The renoprotective properties of some currently available drugs might be attributable in part to inhibition of Wnt signaling. The development of orally active Wnt modulators will provide a potentially important pharmacological tool for further investigating the role of Wnt/beta-catenin signaling and might offer a novel therapeutic strategy in renal fibrosis.