Role of GD2 and its biosynthetic enzyme GD3 synthase in prostate cancer tumorigenesis.

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel, targetable, pathways that contribute to tumor progression of PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC has been only little explored. Here, we show that GD2 is expressed on a small subpopulation of PC cells in a subset of patients, especially in metastatic PC. Variable levels of cell surface GD2 expression are seen in most PC cell lines, and the expression is highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction is enriched upon growth of PC cells as tumorspheres and GD2high fraction is enriched in tumorsphere growth. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2-high CRPC cell models led to marked impairment of their in vitro oncogenic traits, reduced cancer stem cell (CSC) and epithelial-mesenchymal transition (EMT) marker expression and growth as bone-implanted xenograft tumors. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

Understanding the role of Pax5 in development of taxane-resistant neuroendocrine like prostate cancers.

Resistance to the current Androgen Receptor Signaling Inhibitor (ARSI) therapies has led to higher incidences of therapy-induced neuroendocrine-like prostate cancer (t-NEPC). This highly aggressive subtype with predominant small cell-like characteristics is resistant to taxane chemotherapies and has a dismal overall survival. t-NEPCs are mostly treated with platinum-based drugs with a combination of etoposide or taxane and have less selectivity and high systemic toxicity, which often limit their clinical potential. During t-NEPC transformation, adenocarcinomas lose their luminal features and adopt neuro-basal characteristics. Whether the adaptive neuronal characteristics of t-NEPC are responsible for such taxane resistance remains unknown. Pathway analysis from patient gene-expression databases indicates that t-NEPC upregulates various neuronal pathways associated with enhanced cellular networks. To identify transcription factor(s) (TF) that could be important for promoting the gene expression for neuronal characters in t-NEPC, we performed ATAC-Seq, acetylated-histone ChIP-seq, and RNA-seq in our NE-like cell line models and analyzed the promoters of transcriptionally active and significantly enriched neuroendocrine-like (NE-like) cancer-specific genes. Our results indicate that Pax5 could be an important transcription factor for neuronal gene expression and specific to t-NEPC. Pathway analysis revealed that Pax5 expression is involved in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are critical for strong cellular communications. Further results suggest that depletion of Pax5 disrupts cellular interaction in NE-like cells and reduces surface growth factor receptor activation, thereby, sensitizing them to taxane therapies. Moreover, t-NEPC specific hydroxymethylation of Pax5 promoter CpG islands favors Pbx1 binding to induce Pax5 expression. Based on our study, we concluded that continuous exposure to ARSI therapies leads to epigenetic modifications and Pax5 activation in t-NEPC, which promotes the expression of genes necessary to adopt taxane-resistant NE-like cancer. Thus, targeting the Pax5 axis can be beneficial for reverting their taxane sensitivity.

Role of Neuropilin-2-mediated signaling axis in cancer progression and therapy resistance.

Neuropilins (NRPs) are transmembrane proteins involved in vascular and nervous system development by regulating angiogenesis and axon guidance cues. Several published reports have established their role in tumorigenesis. NRPs are detectable in tumor cells of several cancer types and participate in cancer progression. NRP2 is also expressed in endothelial and immune cells in the tumor microenvironment and promotes functions such as lymphangiogenesis and immune suppression important for cancer progression. In this review, we have taken a comprehensive approach to discussing various aspects of NRP2-signaling in cancer, including its regulation, functional significance in cancer progression, and how we could utilize our current knowledge to advance the studies and target NRP2 to develop effective cancer therapies.

Neuropilin-2 axis in regulating secretory phenotype of neuroendocrine-like prostate cancer cells and its implication in therapy resistance.

Neuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2's role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy.

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Prostate cancer addiction to oxidative stress defines sensitivity to anti-tumor neutrophils.

Bone metastatic prostate cancer (BM-PCa) remains one of the most difficult cancers to treat due to the complex interactions of cancer and stromal cells. We previously showed that bone marrow neutrophils elicit an anti-tumor immune response against BM-PCa. Further, we demonstrated that BM-PCa induces neutrophil oxidative burst, which has previously been identified to promote primary tumor growth of other cancers, and a goal of this study was to define the importance of neutrophil oxidative burst in BM-PCa. To do this, we first examined the impact of depletion of reactive oxygen species (ROS), via systemic deletion of the main source of ROS in phagocytes, NADPH oxidase (Nox)2, which we found to suppress prostate tumor growth in bone. Further, using pharmacologic ROS inhibitors and Nox2-null neutrophils, we found that ROS depletion specifically suppresses growth of androgen-insensitive prostate cancer cells. Upon closer examination using bulk RNA sequencing analysis, we identified that metastatic prostate cancer induces neutrophil transcriptomic changes that activates pathways associated with response to oxidative stress. In tandem, prostate cancer cells resist neutrophil anti-tumor response via extracellular (i.e., regulation of neutrophils) and intracellular alterations of glutathione synthesis, the most potent cellular antioxidant. These findings demonstrate that BM-PCa thrive under oxidative stress conditions and such that regulation of ROS and glutathione programming could be leveraged for targeting of BM-PCa progression.

SUMO Modification of PAF1/PD2 Enables PML Interaction and Promotes Radiation Resistance in Pancreatic Ductal Adenocarcinoma.

RNA polymerase II-associated factor 1 (PAF1)/pancreatic differentiation 2 (PD2) is a core subunit of the human PAF1 complex (PAF1C) that regulates the RNA polymerase II function during transcriptional elongation. PAF1/PD2 has also been linked to the oncogenesis of pancreatic ductal adenocarcinoma (PDAC). Here, we report that PAF1/PD2 undergoes posttranslational modification (PTM) through SUMOylation, enhancing the radiation resistance of PDAC cells. We identified that PAF1/PD2 is preferentially modified by small ubiquitin-related modifier 1 (SUMO 1), and mutating the residues (K)-150 and 154 by site-directed mutagenesis reduces the SUMOylation. Interestingly, PAF1/PD2 was found to directly interact with the promyelocytic leukemia (PML) protein in response to radiation, and inhibition of PAF1/PD2 SUMOylation at K-150/154 affects its interaction with PML. Our results demonstrate that SUMOylation of PAF1/PD2 increased in the radiated pancreatic cancer cells. Furthermore, inhibition of SUMOylation or PML reduces the cell growth and proliferation of PDAC cells after radiation treatment. These results suggest that SUMOylation of PAF1/PD2 interacts with PTM for PDAC cell survival. Furthermore, abolishing the SUMOylation in PDAC cells enhances the effectiveness of radiotherapy. Overall, our results demonstrate a novel PTM and PAF1/PD2 interaction through SUMOylation, and inhibiting the SUMOylation of PAF1/PD2 enhance the therapeutic efficacy for PDAC.

Tumor- and osteoclast-derived NRP2 in prostate cancer bone metastases.

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis.

Neuropilin-2 and Its Transcript Variants Correlate with Clinical Outcome in Bladder Cancer.

Urothelial bladder cancer ranks among the 10 most frequently diagnosed cancers worldwide. In our previous study, the transmembrane protein neuropilin-2 (NRP2) emerged as a predictive marker in patients with bladder cancer. NRP2 consists of several splice variants; the most abundant of these, NRP2a and NRP2b, are reported to have different biological functions in lung cancer progression. For other cancer types, there are no published data on the role of these transcript variants in cancer progression and the clinical outcome. Here, we correlate NRP2 and its two most abundant transcript variants, NRP2A and NRP2B, with the clinical outcome using available genomic data with subsequent validation in our own cohort of patients with muscle-invasive bladder cancer. In addition to NRP2, NRP1 and the NRP ligands PDGFC and PDGFD were studied. Only NRP2A emerged as an independent prognostic marker for shorter cancer-specific survival in muscle-invasive bladder cancer in our cohort of 102 patients who underwent radical cystectomy between 2008 and 2014 with a median follow-up time of 82 months. Additionally, we demonstrate that high messenger expression of NRP2, NRP1, PDGFC and PDGFD associates with a more aggressive disease (i.e., a high T stage, positive lymph node status and reduced survival).

Deubiquitination in prostate cancer progression: role of USP22.

Prostate cancer (PCa) is the leading cause of cancer death in men. With more therapeutic modalities available, the overall survival in PCa has increased significantly in recent years. Patients with relapses after advanced secondgeneration anti-androgen therapy however, often show poor disease prognosis. This group of patients often die from cancer-related complicacies. Multiple approaches have been taken to understand disease recurrence and to correlate the gene expression profile. In one such study, an 11-gene signature was identified to be associated with PCa recurrence and poor survival. Amongst them, a specific deubiquitinase called ubiquitin-specific peptidase 22 (USP22) was selectively and progressively overexpressed with PCa progression. Subsequently, it was shown to regulate androgen receptors and Myc, the two most important regulators of PCa progression. Furthermore, USP22 has been shown to be associated with the development of therapy resistant PCa. Inhibiting USP22 was also found to be therapeutically advantageous, especially in clinically challenging and advanced PCa. This review provides an update of USP22 related functions and challenges associated with PCa research and explains why targeting this axis is beneficial for PCa relapse cases.

Macrophage-Derived Neuropilin-2 Exhibits Novel Tumor-Promoting Functions.

Tumor-associated macrophages (TAM) are causally associated with tumorigenesis as well as regulation of antitumor immune responses and have emerged as potential immunotherapeutic targets. Recent evidence suggests TAM phagocytose apoptotic tumor cells within the tumor microenvironment through efferocytosis in an immunologically silent manner, thus maintaining an immunosuppressed microenvironment. The signal transduction pathways coupling efferocytosis and immunosuppression are not well known. Neuropilin-2 (NRP2) is a member of the membrane-associated neuropilin family and has been reported in different immune cells but is poorly characterized. In this study, we show that NRP2 is expressed during macrophage differentiation, is induced by tumor cells, and regulates phagocytosis in macrophages. Furthermore, NRP2 in TAM promoted efferocytosis and facilitated tumor growth. Deletion of NRP2 from TAM impaired the clearance of apoptotic tumor cells and increased secondary necrosis within tumors. This resulted in a break in the immune tolerance and reinitiated antitumor immune responses, characterized by robust infiltration of CD8+ T and natural killer cells. This result suggests NRP2 may act as a molecular mediator that connects efferocytosis and immune suppression. Deletion of NRP2 in TAM downregulated several immunosuppressive and tumor-promoting genes and upregulated immunostimulatory genes in the myeloid compartment. Taken together, our study demonstrates that TAM-derived NRP2 plays a crucial role in tumor promotion through efferocytosis, opening the enticing option for the development of effective immunotherapy targeting TAM.Significance: Neuropilin-2 in macrophages promotes tumor growth by regulating efferocytosis of apoptotic tumor cells and orchestrating immune suppression.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/19/5600/F1.large.jpg Cancer Res; 78(19); 5600-17. ©2018 AACR.

Hyaluronic acid formulation of near infrared fluorophores optimizes surgical imaging in a prostate tumor xenograft.

The presence of positive surgical margins confers an increased risk of biochemical relapse and need for salvage therapy in men undergoing radical prostatectomy. Image-guided surgery using near-infrared (NIR) fluorescent contrast agents is a potential method to detect remaining cancerous tissue. The objective of this study was to evaluate three hyaluronic acid (HA) nanoparticle (NP) formulations loaded with NIR fluorophore for their ability to contrast-enhance prostate cancer. HA was modified by conjugation with the hydrophobic ligand, aminopropyl-1-pyrenebutanamide to drive nanoparticle self-assembly. Indocyanine green (ICG) was physicochemically entrapped in the HA-NP, termed NanoICG. Alternatively, Cy7.5 was directly conjugated to amphiphilic HA, termed NanoCy7.5. NanoCy7.5 was synthesized with two HA molecular weights to determine the HA size contribution to delivery to PC3 prostate tumor xenografts. Contrast-enhancement of the tumors and relative biodistribution were assessed by a series of fluorescence imaging, image-guided surgery with spectroscopy, and microscopic techniques. Intravenously administered NanoICG improved tumor signal-to-noise ratio (SNR) at 24 h over ICG by 2.9-fold. NanoCy7.5 with 10 kDa and 100 kDa HA improved tumor SNR by 6.6- and 3.1-fold over Cy7.5 alone, respectively. The PC3 xenograft was clearly identified with the image-guided system providing increased contrast enhancement compared to surrounding tissue for NanoICG and NanoCy7.5 with 10 kDa HA. NIR fluorescence microscopy showed that Cy7.5 in NPs with 10 kDa HA were distributed throughout the tumor, while NanoCy7.5 with 100 kDa HA or NanoICG delivered dye mainly to the edge of the tumor. CD31 staining suggested that PC3 tumors are poorly vascularized. These studies demonstrate the efficacy of a panel of HA-derived NPs in identifying prostate tumors in vivo, and suggest that by tuning the structural properties of these NPs, optimized delivery can be achieved to poorly vascularized tumors. STATEMENT OF SIGNIFICANCE: We have demonstrated the potential of a panel of near-infrared fluorescent (NIRF) nanoparticles (NPs) for image-guided surgery in a prostate cancer xenograft model. Image-guided surgery and imaging of organs ex vivo showed greater tumor signal and contrast when mice were administered NIRF dyes that were covalently conjugated to (NanoCy7.510k-PBA) or physicochemically entrapped in (NanoICGPBA) hyaluronic acid (HA) NPs, compared to free dyes. These results show the potential to use these NPs as tools to detect the margins of tumors and to differentiate healthy and tumor tissue intraoperatively. Moreover, this project provides insight into selecting optimal formulation strategies for poorly vascularized tumors.

Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma.

DNA methyltransferase 3A (DNMT3A) is an enzyme involved in DNA methylation that is frequently mutated in human hematologic malignancies. We have previously shown that inactivation of Dnmt3a in hematopoietic cells results in chronic lymphocytic leukemia in mice. Here we show that 12% of Dnmt3a-deficient mice develop CD8+ mature peripheral T cell lymphomas (PTCL) and 29% of mice are affected by both diseases. 10% of Dnmt3a+/- mice develop lymphomas, suggesting that Dnmt3a is a haploinsufficient tumor suppressor in PTCL. DNA methylation was deregulated genome-wide with 10-fold more hypo- than hypermethylated promoters and enhancers, demonstrating that hypomethylation is a major event in the development of PTCL. Hypomethylated promoters were enriched for binding sites of transcription factors AML1, NF-κB and OCT1, implying the transcription factors potential involvement in Dnmt3a-associated methylation. Whereas 71 hypomethylated genes showed an increased expression in PTCL, only 3 hypermethylated genes were silenced, suggesting that cancer-specific hypomethylation has broader effects on the transcriptome of cancer cells than hypermethylation. Interestingly, transcriptomes of Dnmt3a+/- and Dnmt3aΔ/Δ lymphomas were largely conserved and significantly overlapped with those of human tumors. Importantly, we observed downregulation of tumor suppressor p53 in Dnmt3a+/- and Dnmt3aΔ/Δ lymphomas as well as in pre-tumor thymocytes from 9 months old but not 6 weeks old Dnmt3a+/- tumor-free mice, suggesting that p53 downregulation is chronologically an intermediate event in tumorigenesis. Decrease in p53 is likely an important event in tumorigenesis because its overexpression inhibited proliferation in mouse PTCL cell lines, suggesting that low levels of p53 are important for tumor maintenance. Altogether, our data link the haploinsufficient tumor suppressor function of Dnmt3a in the prevention of mouse mature CD8+ PTCL indirectly to a bona fide tumor suppressor of T cell malignancies p53.

NRP2 transcriptionally regulates its downstream effector WDFY1.

Neuropilins (NRPs) are cell surface glycoproteins that often act as co-receptors for plexins and VEGF family receptors. Neuropilin-2 (NRP2), a family member of NRPs, was shown to regulate autophagy and endocytic trafficking in cancer cells, a function distinctly different from its role as a co-receptor. WD Repeat and FYVE domain containing 1 (WDFY1)-protein acts downstream of NRP2 for this function. Our results indicated that NRP2 maintains an optimum concentration of WDFY1 by negatively regulating its expression. Since increased expression of WDFY1 reduces the endocytic activity, maintenance of WDFY1 level is crucial in metastatic cancer cells to sustain high endocytic activity, essential for promotion of oncogenic activation and cancer cell survival. Here, we have delineated the underlying molecular mechanism of WDFY1 synthesis by NRP2. Our results indicated that NRP2 inhibits WDFY1 transcription by preventing the nuclear localization of a transcription factor, Fetal ALZ50-reactive clone 1 (FAC1). Our finding is novel as transcriptional regulation of a gene by NRP2 axis has not been reported previously. Regulation of WDFY1 transcription by NRP2 axis is a critical event in maintaining metastatic phenotype in cancer cells. Thus, inhibiting NRP2 or hyper-activating WDFY1 can be an effective strategy to induce cell death in metastatic cancer.

Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development.

Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

Neuropilin-2 Regulates Endosome Maturation and EGFR Trafficking to Support Cancer Cell Pathobiology.

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies, where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface EGFR, arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat- and FYVE-domain-containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies.

Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis.

DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3a(Δ/Δ) mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3a(Δ/Δ) lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3a(Δ/Δ) mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.

Upregulation of dendritic arborization by N-acetyl-D-glucosamine kinase is not dependent on its kinase activity.

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the γ-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.