The PIAS-like Coactivator Zmiz1 Is a Direct and Selective Cofactor of Notch1 in T Cell Development and Leukemia

SummaryPan-NOTCH inhibitors are poorly tolerated in clinical trials because NOTCH signals are crucial for intestinal homeostasis. These inhibitors might also promote cancer because NOTCH can act as a tumor suppressor. We previously reported that the PIAS-like coactivator ZMIZ1 is frequently co-expressed with activated NOTCH1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we show that similar to Notch1, Zmiz1 was important for T cell development and controlled the expression of certain Notch target genes, such as Myc. However, unlike Notch, Zmiz1 had no major role in intestinal homeostasis or myeloid suppression. Deletion of Zmiz1 impaired the initiation and maintenance of Notch-induced T-ALL. Zmiz1 directly interacted with Notch1 via a tetratricopeptide repeat domain at a special class of Notch-regulatory sites. In contrast to the Notch cofactor Maml, which is nonselective, Zmiz1 was selective. Thus, targeting the NOTCH1-ZMIZ1 interaction might combat leukemic growth while avoiding the intolerable toxicities of NOTCH inhibitors

Periostin Responds to Mechanical Stress and Tension by Activating the MTOR Signaling Pathway

Current knowledge about Periostin biology has expanded from its recognized functions in embryogenesis and bone metabolism to its roles in tissue repair and remodeling and its clinical implications in cancer. Emerging evidence suggests that Periostin plays a critical role in the mechanism of wound healing; however, the paracrine effect of Periostin in epithelial cell biology is still poorly understood. We found that epithelial cells are capable of producing endogenous Periostin that, unlike mesenchymal cell, cannot be secreted. Epithelial cells responded to Periostin paracrine stimuli by enhancing cellular migration and proliferation and by activating the mTOR signaling pathway. Interestingly, biomechanical stimulation of epithelial cells, which simulates tension forces that occur during initial steps of tissue healing, induced Periostin production and mTOR activation. The molecular association of Periostin and mTOR signaling was further dissected by administering rapamycin, a selective pharmacological inhibitor of mTOR, and by disruption of Raptor and Rictor scaffold proteins implicated in the regulation of mTORC1 and mTORC2 complex assembly. Both strategies resulted in ablation of Periostin-induced mitogenic and migratory activity. These results indicate that Periostin-induced epithelial migration and proliferation requires mTOR signaling. Collectively, our findings identify Periostin as a mechanical stress responsive molecule that is primarily secreted by fibroblasts during wound healing and expressed endogenously in epithelial cells resulting in the control of cellular physiology through a mechanism mediated by the mTOR signaling cascade.This work was funded by the National Institutes of Health (NIH/NCI) P50-CA97248 (University of Michigan Head and Neck SPORE)

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Sequential eukaryotic translation initiation factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2β stabilizes the 48S preinitiation complex and promotes its shift to the initiation mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eIF4G and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1 and mRNA. Here we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with eIF5-C-terminal domain (CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro, in a manner reversed by eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes and eIF5-CTD restore the accuracy of start codon selection impaired by a eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start-codon-induced release of eIF1, the major antagonist of establishing tRNA[subscript i][superscript Met]:mRNA binding to the P-site

Periostin-driven cellular migration requires mTOR signaling.

<p>(<b>A</b>) Representative pictures of the NOK-SI cell scratch assay following treatment with vehicle, recombinant Periostin (50 ng/ml), and rapamycin (50 nM). Scale bars represent 50 μm. (<b>B</b>) Quantitative analysis of open-wounded area over time (n=4; mean ± S.E.M.). Note that rapamycin abrogates the Periostin migratory activity of epithelial cells (***p<0.001). (<b>C</b>) Proliferation assay using keratinocytes treated with rapamycin and/or Periostin. Note that Periostin alone induced significant cellular proliferation at 50 ng/ml (*p<0.05). Treatment with rapamycin blocked periostin-induced cell proliferation (ns p>0.05). (<b>D</b>) Representative immunoblot depicting knockdown of Raptor and Rictor after siRNA treatment. Scramble siRNA oligonucleotides sequences were used as controls. GAPDH was used as loading controls. (<b>E</b>) Graphic shows the quantitative analyses of open-wounded areas using NOK-SI cells over time (n=4; mean ± S.E.M.). Note that siRNA targeting Raptor abrogates Periostin-induced cellular migratory resulting on complete wound closure by 48 hours (**p<0.05). siRNA targeting Rictor did not change the Periostin induced accelerated cellular migration resulting on wound closure by 24 hours (ns p>0.05). (<b>F</b>) Proliferation assay using NOK-SI cells treated with siRNA for Raptor, Rictor, or siRNA scramble, and/or Periostin. Note that Periostin induced significant cellular proliferation (*p<0.05). Treatment with siRNA for Raptor or Rictor resulted in disruption of Periostin induced cellular proliferation (***p<0.001).</p

Co-expression of Periostin and mTOR during cellular migration and mechanical stress induced by tension.

<p>(<b>A</b>) A representative wound healing scratch assay shows keratinocytes stained for Periostin (TRITC-red), pS6 (FITC-green) and DNA (Hoechst-blue). Note colocalization of Periostin and pS6 staining (on merge and insert) in the migratory area. Scale bars represent 50 μm. (<b>B</b>) Quantification of positive cells co-expressing Periostin and pS6 are depicted. Note increased number of positive cells co-expressing Periostin and pS6. Most of these cells are in the migratory area (***p<0.001). (<b>C</b>) NOK-SI cells were subjected to biomechanical stimulation (load of 14% stretching at 6 cycles/min) at the indicated time points. (<b>D</b>) Western blot analysis for Periostin and pS6 expression in NOK-SI subjected to load assay. Non-stimulated cells (no load force) served as a control, and GAPDH was used as protein loading control.</p

F-actin polarization and PI3K/mTOR signaling activation by Periostin-induced epithelial cell migration.

<p>(<b>A</b>) Phalloidin detection shows cells with polarized F-actin (white arrow) following treatment with recombinant Periostin compared to vehicle control. Scale bars represent 10 μm. (<b>B</b>) Graphic represents percentage of cells with stress fiber formation (polarized F-actin) after periostin or vehicle stimuli. Results were determined by measuring fields using independent triplicates (**p<0.01) (<b>C</b>) Activation of PI3K and mTOR signaling is triggered by Periostin treatment in a dose-dependent manner, as detected by phosphorylated AKT at Threonine 308 (pAKT^Thr308) and Serine 473 (pAKT^Ser473) and phosphorylated S6 (pS6). Note that 50 ng/ml of Periostin is the optimal concentration for PI3K activation. GAPDH was used as a loading control. </p

Periostin-driven migration and proliferation.

<p>(<b>A</b>) Total cell lysates and conditioned medium (cond. medium) from NOK-SI and hPDL cells were blotted for Periostin. New cell culture medium supplemented with 10% FBS was used as a negative control (control). Intracellular Periostin is detected in epithelial cell lysate. However, conditioned medium from NOK-SI shows that keratinocytes did not secrete Periostin, as the same band was observed in the negative control media. hPDL cells have low levels of the intracellular Periostin isoform as observed in the cell lysate. Increased levels of secreted Periostin were found in the conditioned medium from hPDL. (<b>B</b>) hPDL conditioned medium induces keratinocyte proliferation compared to vehicle alone (***p<0.001), which is reduced upon administration of anti-Periostin antibody (*p<0.05). (<b>C</b>) Representative pictures of NOK-SI migration following treatment with recombinant Periostin (50 ng/ml), EGF (100 ng/ml) as the positive control, or vehicle. Scale bars represent 50 μm. (<b>D</b>) Graphic represents the quantification of the wound areas at indicated times (n=4; mean ± S.E.M). (<b>E</b>) Periostin enhances proliferation of keratinocytes compared to vehicle treated cells (***p<0.001). EGF treatment was used as positive control (*p<0.05) (n=6; mean ± S.E.M).</p

Expression of Periostin and CK6 during cutaneous wound healing.

<p><b>H</b>&<b>E</b>: Representative histological sections of cutaneous incisional wounds. (<b>A</b>) Morphology of the wounded site shows a thin edge of epithelial cells migrating across the wound bed, termed the epithelial tongue and (<b>B</b>) intact and normal skin adjacent to the wounded site were stained with Hematoxylin and Eosin (H&E). (<b>C</b>) Epithelial cells at the epithelial tongue express intracellular Periostin. (<b>D</b>) In normal adjacent skin, Periostin is in the connective tissue at the basal lamina, which is juxtaposed to the epithelial basal layer. (<b>E</b>) Note that basal and parabasal layers of the epithelial tongue have a large number of proliferating BrDU positive cells. (<b>F</b>) As expected, the epithelial basal layer of adjacent skin has few proliferating cells. (<b>G</b>) Upregulation of the epithelia stress/tension marker CK6 is depicted in the epithelial tongue compared to normal adjacent skin observed in (<b>H</b>). Scale bars represent 50 μm.</p