TRF2 promotes,remodels and protects telomeric Holliday junctions

The ability of the telomeric DNA‐binding protein, TRF2, to stimulate t‐loop formation while preventing t‐loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t‐loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t‐loops and at regressed replication forks.     

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.     

Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile

Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.     

Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors

ALK is a receptor tyrosine kinase with an oncogenic role in various types of human malignancies. Despite constitutive activation of the kinase through gene alterations, such as chromosomal translocation, gene amplification or mutation, treatments with kinase inhibitors invariably lead to the development of resistance. Aiming to develop new tools for ALK targeting, we took advantage of our previous demonstration identifying ALK as a dependence receptor, implying that in the absence of ligand the kinase-inactive ALK triggers or enhances apoptosis. Here, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large-cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore, siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore, ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors.Designing targeted therapy for cancer has been a major goal of the last decade. Oncogenic tyrosine kinases have raised early interest, because elucidation of their structure facilitated the development of small-molecule inhibitors with therapeutic efficiency.¹ The pioneer BCR-ABL inhibitor molecule imatinib was approved for therapeutic use as early as 2001 to treat chronic myeloid leukemia and Ph1-positive acute lymphoblastic leukemia.² Later on, inhibitors targeting receptors for epidermal growth factor or vascular endothelial growth factor were approved for treatment of solid tumors, such as lung and breast cancer. To date, many tyrosine kinase inhibitors (TKIs) are used in the clinic.³ However, cancers treated by TKIs invariably become resistant to therapy and relapse. Acquired resistance develops through various mechanisms including secondary mutations of the targeted oncogene or activation of alternative proliferative signaling pathways.⁴ It seems thus necessary to invent new strategies designed to attack the tumor on multiple fronts.ALK (anaplastic lymphoma kinase) is an oncogenic receptor tyrosine kinase associated with many tumor types. ALK was first identified in 1994 as a rearranged gene fusion (NPM–ALK) resulting from the t(2;5)(p23;q35) translocation occurring in 75% human anaplastic large-cell lymphomas (ALCLs).^{5, 6} Other translocations or gene inversions involving ALK were later described in solid tumors including 50–60% inflammatory myofibroblastic tumors, and a small proportion of diffuse large B-cell lymphomas, breast and renal carcinomas.^{7, 8} Recently, 4–8% non-small-cell lung cancer (NSCLC) were found to harbor an echinoderm microtubule-associated protein-like 4 (EML4)–ALK fusion.^{7, 9} Resulting fusion proteins associate the N-terminal portion of a protein partner (containing in most cases a dimerization domain) to the entire intracellular portion of ALK, including its tyrosine kinase domain. Subsequent dimerization of this fusion protein leads to constitutive activation of ALK kinase, resulting in enhanced signaling for cell proliferation, survival and oncogenicity.¹⁰The full-length ALK receptor cDNA codes for a transmembrane receptor tyrosine kinase of the insulin receptor superfamily, which is essentially expressed in the developing nervous system.^{11, 12} Some authors proposed the two heparin-binding factors pleiotrophin (PTN) and midkine as ligands for ALK.¹⁰ However, their binding to ALK is controversed and might be indirectly mediated by heparin.¹³ ALK kinase signaling most likely involves co-receptors and/or co-signaling molecules such as the transmembrane receptor tyrosine phosphatase beta/zeta (RPTPb/z), a receptor for PTN and midkine. In the absence of ligand, RPTPb/z dephosphorylates ALK, whereas PTN and midkine direct binding to RPTPb/z inactivates its phosphatase activity.¹⁴ Expression of the full-length ALK receptor was also observed in neuroblastoma, a pediatric tumor derived from the neural crest affecting the peripheral nervous system. The ALK kinase in neuroblastoma is most often constitutively active as a result of gain-of-function mutations or protein overexpression, due to ALK gene amplification or copy number increase.^{10, 15}ALK appears therefore as an interesting therapeutic target to treat ALK-positive tumors. Indeed, since the identification of NPM–ALK and other ALK fusions as oncogenes for ALCL and inflammatory myofibroblastic tumors,^{6, 16, 17} several pharmaceutical companies developed ALK-specific TKIs. In 2010, a TKI targeting ALK and c-MET, crizotinib¹⁸ (also called PF-02341066), was authorized in clinical trials as a second-line therapy for advanced stage NSCLC harboring EML4–ALK. The initial clinical responses were so encouraging that crizotinib is currently tested in a growing number of advanced ALK-positive tumors (clinicaltrials.gov). Nevertheless, the tumors invariably develop resistance to the inhibitor, mostly through mutations of the kinase active site.^{19, 20} Therefore, it appears necessary to design alternate treatments or to associate TKIs with other molecules. One promising strategy would be to impair distinct functions of the oncogenic tyrosine kinase through targeting different sites of the ALK protein.We recently demonstrated that the ALK receptor tyrosine kinase belongs to the functional family of so-called ‘dependence receptors''.^{21, 22} Such dependence receptors function with a dual signaling: in the presence of ligand (or a situation mimicking a ligand, e.g., inducing receptor dimerization and activation), the receptor exerts a prosurvival/antiapoptotic effect on the cell; in contrast, in absence of ligand and when the cell is submitted to environmental or genotoxic stress, a dependence receptor becomes proapoptotic. The proapoptotic effect is mediated by caspase-dependent cleavage of the receptor, either releasing or exposing a proapoptotic domain/sequence (called ‘addiction/dependence domain'' or ADD), thus amplifying the apoptotic process.²³ Molecular analysis of ALK deletion mutants allowed us to map the ADD domain of ALK to a 36-amino-acid (aa) stretch located within the juxtamembrane intracytoplasmic region of ALK. The ADD of ALK lacks homology with any known protein motif implicated in apoptotic processes and is necessary for ALK proapoptotic function.²² The purpose of the present study was to design a novel targeted therapy, taking advantage of the proapoptotic function of ALK.Our hypothesis was that a synthetic peptide could mimic the proapoptotic function of ALK. Therefore, we synthesized several peptides whose sequence reproduced the entire ADD domain (36 aa) of ALK or part of it (12 aa) to assay their effects on various tumor cell lines. We show that several of these ALK-derived peptides are proapoptotic for ALK-expressing, but not ALK-negative, tumor cells. In addition, the ALK-derived 36-aa peptide (P36) enhanced the cytotoxic effect of the ALK kinase inhibitor crizotinib in ALK-positive ALCL and neuroblastoma cell lines. Thus our results uncover a new strategy for targeting ALK-expressing tumors.     

Effortless Attention as a Biomarker for Experienced Mindfulness Practitioners

Objective

The present study aimed at comparing frontal beta power between long-term (LTM) and first-time meditators (FTM), before, during and after a meditation session. We hypothesized that LTM would present lower beta power than FTM due to lower effort of attention and awareness.

Methods

Twenty one participants were recruited, eleven of whom were long-term meditators. The subjects were asked to rest for 4 minutes before and after open monitoring (OM) meditation (40 minutes).

Results

The two-way ANOVA revealed an interaction between the group and moment factors for the Fp1 (p<0.01), F7 (p = 0.01), F3 (p<0.01), Fz (p<0.01), F4 (p<0.01), F8 (p<0.01) electrodes.

Conclusion

We found low power frontal beta activity for LTM during the task and this may be associated with the fact that OM is related to bottom-up pathways that are not present in FTM.

Significance

We hypothesized that the frontal beta power pattern may be a biomarker for LTM. It may also be related to improving an attentive state and to the efficiency of cognitive functions, as well as to the long-term experience with meditation (i.e., life-time experience and frequency of practice).