Dynamin-2 Expression Research Articles

Abstract 11484: Upregulation of Dynamin 2 Increases Mitochondrial Fission and Cell Proliferation in Pulmonary Arterial Hypertension

Introduction: The obstructive vasculopathy in pulmonary arterial hypertension (PAH) results in part from acquired mitochondrial changes in pulmonary arterial smooth muscle cells (PASMC), including increased mitotic fission. Mitotic fission, mitochondrial division which is coordinated with mitosis to equitably distribute mitochondria to daughter cells, is mediated by activation of dynamin-related protein 1 (Drp1). To complete mitochondrial fission, Drp1 requires assistance from the GTPase dynamin 2 (DNM2). Hypothesis: Pathological increase in DNM2 expression drives mitochondrial fission and proliferation of PAH PASMC. Methods: Immunoblots of human PASMC and immunohistochemistry and immunofluorescence of lung sections from human (n=5/group) and monocrotaline (MCT) rats with PAH (n=4-5/group) were used to assess the expression of DNM2. The effect of manipulating DNM2 on cell proliferation, cell cycle and apoptosis were assessed by flow cytometry. Mitochondrial fission was quantified using confocal imaging. Results: DNM2 protein expression is increased in human PAH PASMC. DNM2 is also increased in the media of small pulmonary arteries of PAH patients and MCT-PAH rats. Silencing DNM2 inhibited mitochondrial fission and reduced proliferation causing cell cycle arrest in G1/G0 phase. Silencing DNM2 in PAH PASMC also induced apoptosis. Conversely, augmenting DNM2 in normal PASMC induced mitochondrial fission and accelerated cell proliferation. miR-124-3p, a putative negative regulator of DNM2, is decreased in PAH PASMC. Augmenting miR-124-3p decreased DNM2 expression, inhibited proliferation and induced apoptosis in PAH PASMC. Conclusion: In health, DNM2 regulates mitochondrial fission whilst in disease, epigenetic upregulation of DNM2 increases mitotic fission, which drives pathologic proliferation and apoptosis resistance. The miR-124-3p-DNM2 pathway offers novel PAH therapeutic targets.

Overexpression of cytoplasmic dynamin 2 is associated with worse outcomes in patients with clear cell renal cell carcinoma.

Dynamin 2 (DNM2) involved in tumor progression in various malignancies. For the first time, we evaluated DNM2 expression pattern, its association with clinicopathological characteristics and survival outcomes in RCC subtypes. We evaluated the DNM2 expression pattern in RCC tissues as well as adjacent normal tissue using immunohistochemistry on tissue microarray (TMA) slides. Our findings revealed increased DNM2 expression in RCC samples rather than in adjacent normal tissues. The results indicated that there was a statistically significant difference between cytoplasmic expression of DNM2 among subtypes of RCC in terms of intensity of staining, percentage of positive tumor cells, and H-score (P= 0.024, 0.049, and 0.009, respectively). The analysis revealed that increased cytoplasmic expression of DNM2 in ccRCC is associated with worse OS (log rank: P= 0.045), DSS (P= 0.049), and PFS (P= 0.041). Furthermore, cytoplasmic expression of DNM2 was found as an independent prognostic factor affecting DSS and PFS in multivariate analysis. Our results indicated that DNM2 cytoplasmic expression is associated with tumor aggressiveness and poor outcomes. DNM2 could serve as a promising prognostic biomarker and therapeutic target in patients with ccRCC.

Oxidative stress regulates cardiomyocyte energy metabolism through the IGF2BP2-dynamin2 signaling pathway

BackgroundOver the past few decades, numerous clinical and experimental studies have confirmed that oxidative stress is enhanced in heart failure (HF). We recently found that inhibition of highly expressed dynamin2 can protect myocardial ischemia-reperfusion injury in mice and inhibit oxidative stress in ischemic cardiomyocytes. However, the specific mechanisms are still not fully understood. In this study, we hypothesized that oxidative stress induces cardiomyocyte apoptosis through IGF2BP2 regulation, which is regulated through the dynamin2 expression. MethodsH2O2-treated cardiomyocytes were observed for the regulatory effect of reactive oxygen species (ROS) on IGF2BP2 and the effect of IGF2BP2 on dynamin2 gene expression was determined by lentiviral-mediated IGF2BP2 overexpression. Then, siRNA knockdown of dynamin2 was used to observe whether it can alter the effect of IGF2BP2 on myocardial cells. Finally, IGF2BP2 was knocked down in ischemic rats using shRNA to determine the effects of IGF2BP2 on myocardial ischemia. ResultsROS can promote dynamin2 expression by inducing IGF2BP2 expression and dynamin2 knockdown could reduce the injury of IGF2BP2 to cardiomyocytes. Inhibition of IGF2BP2 expression in myocardial ischemic rats ameliorated cardiac fibrosis in ischemic myocardium. ConclusionOxidative stress can induce cardiomyocyte apoptosis through the IGF2BP2-dynamin2 pathway. Inhibition of IGF2BP2 expression significantly improves the fibrosis and remodeling that occurs in ischemic myocardium.

Abstract 12932: An Epigenetic Upregulation of Dynamin 2, a Regulator of Mitochondrial Fission, Promotes Human and Experimental Pulmonary Arterial Hypertension

Introduction: Increase in mitotic fission in pulmonary arterial smooth muscle cells (PASMC) contributes to the obstructive vasculopathy in pulmonary arterial hypertension (PAH). PAH’s fissogenic phenotype is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp1). Recent observation suggests that to complete mitochondrial fission, Drp1 requires assistance from the GTPase dynamin 2 (DNM2). In this study we explore the role of DNM2 in the pathogenesis of PAH. Hypothesis: In PAH, increased expression of DNM2 increases mitochondrial fission and proliferation of PASMC. Methods: Immunoblot was used to quantify the expression of DNM2 in control (n=4) and PAH PASMC (n=5). Immunofluorescence was used to quantify the expression of DNM2 in the pulmonary arteries of monocrotaline (MCT) vs control rats. Flow cytometry was used to assess the effects of manipulating the expression of DNM2 on cell proliferation, cell cycle progression and rates of unstimulated apoptosis. Mitochondrial network structure was assessed using confocal imaging of cells infected with Adv-TOM20 mNeon green. Results: The expression of DNM2 was increased in human PAH PASMC vs control PASMC. Increase in the expression of DNM2 expression was also observed in the pulmonary arteries of MCT-PAH rats. Silencing DNM2 inhibited mitochondrial fission in PAH PASMC. Furthermore, silencing DNM2 inhibited proliferation in PAH PASMC by blocking the cell cycle at G1/G0. Conversely, augmenting DNM2 in normal PASMC induced mitochondrial fission and accelerated cell proliferation, recapitulating the PAH phenotype. In silico analysis identified miR-124-3p as a putative negative regulator of DNM2. Augmenting miR-124-3p decreased DNM2 expression, inhibited proliferation and induced apoptosis in PAH PASMC. Conclusion: DNM2 promotes mitochondrial fission and regulates cell proliferation in human PAH PASMC. Decreased miR-124-3p expression may contribute to upregulation of DNM2 in PAH. This work highlights the importance of DNM2 as partner with Drp1 in regulation of mitochondrial fission and shows that like Drp1, DNM2 is increased in human PAH. DNM2 may constitute new therapeutic target for PAH.

Dynamin-2 mediates clathrin-dependent endocytosis for amyloid-β internalization in brain microvascular endothelial cells.

Dynamin is recognized as a crucial regulator for membrane fission and has three isoforms in mammals. But the expression patterns of dynamin isoforms and their roles in non-neuronal cells are incompletely understood. In this study, the expression profiles of dynamin isoforms and their roles in endocytosis was investigated in brain endothelial cells. We found that Dyn2 was expressed at highest levels, whereas the expression of Dyn1 and Dyn3 were far less than Dyn2. Live-cell imaging was used to investigate the effects of siRNA-mediated knockdown of individual dynamin isoforms on transferrin uptake, and we found that Dyn2, but not Dyn1 or Dyn3, is required for the endocytosis in brain endothelial cells. Results of dextran uptake assay showed that dynamin isoforms are not involved in the clathrin-independent fluid-phase internalization of brain endothelial cells, suggesting the specificity of the role of Dyn2 in clathrin-dependent endocytosis. Immunofluorescence and electron microscopy analysis showed that Dyn2 co-localizes with clathrin and acts at the late stage of vesicle fission in the process of endocytosis. Further results showed that Dyn2 is necessary for the basolateral-to-apical internalization of amyloid-β into brain endothelial cells. We concluded that Dyn2, but not Dyn1 or Dyn3, mediates the clathrin-dependent endocytosis for amyloid-β internalization particularly from basolateral to apical side into brain endothelial cells.

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Osteopontin and LDLR Are Upregulated in Hearts of Sudden Cardiac Death Victims With Heart Failure With Preserved Ejection Fraction and Diabetes Mellitus.

Background: Diabetes mellitus (DM) is associated with increased risk of sudden cardiac death (SCD), particularly in patients with heart failure with preserved ejection fraction (HFpEF). However, there are no known biomarkers in the population with DM and HFpEF to predict SCD risk.Objectives: This study was designed to test the hypothesis that osteopontin (OPN) and some proteins previously correlated with OPN, low-density lipoprotein receptor (LDLR), dynamin 2 (DNM2), fibronectin-1 (FN1), and 2-oxoglutarate dehydrogenase-like (OGDHL), are potential risk markers for SCD, and may reflect modifiable molecular pathways in patients with DM and HFpEF.Methods: Heart tissues were obtained at autopsy from 9 SCD victims with DM and HFpEF and 10 age and gender-matched accidental death control subjects from a Finnish SCD registry and analyzed for the expression of OPN and correlated proteins, including LDLR, DNM2, FN1, and OGDHL by immunohistochemistry.Results: We observed a significant upregulation in the expression of OPN, LDLR, and FN1, and a marked downregulation of DNM2 in heart tissues of SCD victims with DM and HFpEF as compared to control subjects (p < 0.01).Conclusions: The dysregulated protein expression of OPN, LDLR, FN1, and DNM2 in patients with DM and HFpEF who experienced SCD provides novel potential modifiable molecular pathways that may be implicated in the pathogenesis of SCD in these patients. Since secreted OPN and soluble LDLR can be measured in plasma, these results support the value of further prospective studies to assess the predictive value of these plasma biomarkers and to determine whether tuning expression levels of OPN and LDLR alters SCD risk in patients with DM and HFpEF.

Dynamin 2 Is Correlated with Recurrence and Poor Prognosis of Papillary Thyroid Cancer.

BackgroundPapillary thyroid cancer (PTC) is the most common histological type of thyroid cancer. Most PTC patients have favorable outcomes, but 10% of patients still have distant metastases at presentation or during follow-up. Dynamin 2 (DNM2) is the only DNM ubiquitously expressed in human tissues, but its expression and clinical significance in PTC is still unknown.Material/MethodsIn our study, we investigated the expression of DNM2 in 112 cases of PTC and classified the patients into low and high expression of DNM2. The clinical significance of DNM2 was evaluated by assessing its correlation with the clinicopathological parameters with the chi-square method. The correlations between DNM2 expression and the disease-free survival rate or overall survival rate were assessed with the Kaplan-Meier method and the log-rank test. The independent prognostic factors of PTC were determined by the Cox-regression hazard model.ResultsPatients with low and high DNM2 expression accounted for 75% and 25% respectively in the 112 patients with PTC. High DNM2 expression was significantly associated with recurrence (P=0.014) and poor prognosis (P=0.004). In addition to tumor stage, DNM2 expression was an independent prognostic biomarker of PTC, indicating an unfavorable prognosis.ConclusionsDNM2 was an independent PTC biomarker indicating more likely recurrence and poorer prognosis. Detecting DNM2 expression may help to select the high-risk patients for adjuvant therapy.

P.104Skeletal muscle reduction of Dnm2 with antisense oligonucleotides in myotubular myopathy

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. The most severe and neonatal X-linked form is known as myotubular myopathy and caused by loss-of-function mutations in Myotubularin (MTM1), while the main autosomal dominant form is due to mutations in Dynamin 2 (DNM2). We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. We are now investigating skeletal muscle targeting of Dnm2 reduction in mice. Indeed specific muscle isoforms are expressed during muscle maturation and in adulthood, through alternative splicing. A single intraperitoneal injection of 25mg/kg of antisense oligonucleotides targeting the total pool of Dnm2 increased the lifespan, whole body strength, and reduced disease severity in Mtm1KO mice, however a single injection alone was not sufficient to rescue the disease for the lifespan of the mice. Repeated injections of ASO was able to revert the disease phenotype in mice within 2 weeks, and the effects lasted several months after the final injection. ASO was still detected in skeletal muscles at this time point, but not in key tissues such as the liver, which may explain in part the longterm improvement in the myopathic phenotype. New ligand-conjugated antisense (LICA) oligonucleotides targeting Dnm2 are now being tested in Mtm1KO mice for potency and efficacy. ASO-mediated Dnm2 knockdown can efficiently correct skeletal muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease. Effective muscle targeting will be an important step in translation of this approach to the clinic for patients.

Caveolin-1 and dynamin-2 overexpression is associated with the progression of bladder cancer.

Caveolae-mediated endocytosis regulates cell adhesion and growth in an anchorage-dependent manner. Studies of the endocytic function of caveolae have suggested a wide-ranging list of cargoes, including a number of receptors and extracellular proteins, ligands and nutrients from the extracellular matrix. Disruption of the processes of caveolae-mediated endocytosis mediated by signaling proteins is critical to cellular integrity. Caveolin-1 and dynamin-2 are the 2 major proteins associated with endocytotic function. Mechanistically, dynamin-2 has a co-equal role with caveolin-1 in terms of caveolae-derived endosome formation. Recent studies have revealed the pathological outcomes associated with the dysregulation of caveolin-1 and dynamin-2 expression. Increased expression levels of the gene for caveolin, Cav-1, resulting in augmented cellular metastasis and invasion, have been demonstrated in various types of cancer, and overexpression of the gene for dynamin-2, DNM2, has been associated with tumorigenesis in cervical, pancreatic and lung cancer. An increased expression of Cav-1 and DNM2 is known to be associated with the invasive behavior of cancer cells, and with cancer progression. Furthermore, it has been previously demonstrated that, in caveolar assembly and caveolae mediated endocytosis, Cav-1 interacts directly with DNM2 during the processes. Altered expression of the 2 genes is critical for the normal function of the cell. The expression patterns of Cav-1 and DNM2 have been previously examined in bladder cancer cell lines, and were each demonstrated to be overexpressed. In the present study, the expression levels of these 2 genes in bladder cancer samples were quantified. The gene expression levels of Cav-1 and DNM2 were identified to be increased 8.88- and 8.62-fold, respectively, in tumors compared with the normal controls. Furthermore, high-grade tumors exhibited significantly increased expression levels of Cav-1 and DNM2 (both P<0.0001) compared with the low-grade tumors. In addition, compared with normal control samples, the expression of the 2 genes in tumor samples was observed to be highly significant (P<0.0001), with a marked positive correlation identified for the tumors (Pearson's correlation coefficient, r=0.80 for the tumor samples vs. r=0.32 in the normal control samples). Taken together, the results of the present study demonstrated that the overexpression of Cav-1 and DNM2 genes, and a determination of their correlation coefficients, may be a potential risk factor for bladder cancer, in addition to other clinical factors.

CONGENITAL MYOPATHIES (CNM): P.143Myostatin as a novel blood based biomarker for antisense oligonucleotide-mediated Dnm2 knockdown to treat myotubular myopathy in mice

Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Most recently we have shown that weekly systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. In addition, systemic weekly ASO injection into severely affected mice leads to reversal of muscle pathology within two weeks. Here we perform a single injection of ASO into 3-week-old Mtm1KO mice, and complete clinical, histological and molecular analysis of the duration of the effects after a single injection. A single injection of 25mg/kg of ASO targeting DNM2 increased the lifespan, whole body strength, and reduced disease severity in Mtm1KO mice compared to untreated controls. Despite these results, a single injection alone was not sufficient to rescue the disease for the lifespan of the mice, suggesting repeated treatments will be required. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease. We next analyzed myostatin levels in muscles and blood of Mtm1KO mice. We show myostatin levels in plasma are significantly reduced in Mtm1KO mice, and that treatment by ASOs targeting DNM2 significantly improves myostatin levels in plasma. In addition plasma myostatin levels inversely correlated with the level of DNM2 mRNA in muscles after injection with ASOs. This provides the first evidence of a blood based biomarker that can be used to monitor disease state and rescue in myotubular myopathy mice. With clinical trials for myotubular myopathy currently in progress, these results are of high relevance to the research field.

Hypoxic stress upregulates Kir2.1 expression by a pathway including hypoxic-inducible factor-1α and dynamin2 in brain capillary endothelial cells.

Brain capillary endothelial cells (BCECs) play a central role in maintenance of blood-brain barrier (BBB) function and, therefore, are essential for central nervous system homeostasis and integrity. Although brain ischemia damages BCECs and causes disruption of BBB, the related influence of hypoxia on BCECs is not well understood. Hypoxic stress can upregulate functional expression of specific K+ currents in endothelial cells, e.g., Kir2.1 channels without any alterations in the mRNA level, in t-BBEC117, a cell line derived from bovine BCECs. The hyperpolarization of membrane potential due to Kir2.1 channel upregulation significantly facilitates cell proliferation. In the present study, the mechanisms underlying the hypoxia-induced Kir2.1 upregulation was examined. We emphasize the involvement of dynamin2, a protein known to be involved in a number of surface expression pathways. Hypoxic culture upregulated dynamin2 expression in t-BBEC117 cells. The inhibition of dynamin2 by Dynasore canceled hypoxia-induced upregulation of Kir2.1 currents by reducing surface expression. On the contrary, Kir2.1 currents and proteins in t-BBEC117 cultured under normoxia were increased by overexpression of dynamin2, but not by dominant-negative dynamin2. Molecular imaging based on bimolecular fluorescence complementation, double-immunostaining, and coimmunoprecipitation assays revealed that dynamin2 can directly bind to the Kir2.1 channel. Moreover, hypoxic culture downregulated hypoxic-inducible factor-1α (HIF-1α) expression. Knockdown of HIF-1α increased dynamin2 expression in t-BBEC117 cells, in both normoxic and hypoxic culture conditions. In summary, our results demonstrated that hypoxia downregulates HIF-1α, increases dynamin2 expression, and facilitates Kir2.1 surface expression, resulting in hyperpolarization of membrane potential and subsequent increase in Ca2+ influx in BCECs.

Intravenous Lipid Emulsion Induces Endocytosis in Human Coronary Artery Endothelial Cells by a CD36/Caveolin‐1 Pathway

IntroductionIntravenous lipid emulsion like Intralipid™ (ILP) are fatty emulsions commonly used in parenteral nutrition. Previous research has shown that they protect against myocardial ischemia/reperfusion injury (IR) in animal models through a mechanism involving activation of Akt/extracellular signal‐regulated kinase‐1/glycogen synthase kinase‐3β pathway ultimately leading to delayed mitochondrial permeability transition pore (mPTP) opening and the induction of a protective effect by ILP. They are also studied in connection with local anesthetic toxicity and mechanisms of scavenging anesthetics from the blood stream.HypothesisThe direct effect of ILP on endothelial cells has not been studied. Hence, the mechanisms by which ILP crosses the endothelium are not well understood especially the effects of ILP on human coronary artery endothelial cells (HCAEC). We hypothesize that uptake of ILP occurs through endocytosis in HCAECs by the formation of a CD36/caveolin‐1 cell membrane complex.MethodsILP in a 1% dilution was administered to HCAECs for 2 hours or control was left untreated. Western blots were conducted with anti‐CD36, caveolin‐1, dynamin‐2, src kinase‐1, eNOS, and phospho‐eNOS antibodies and equal loading of protein confirmed with beta‐actin. Additional cells were exposed to ILP, and then immunohistochemistry was performed, to visualize uptake of ILP into cells. This was accomplished by co‐labeling of lysosomal associated protein 3 CD63 and styryl peptide and lysosomal marker FM5‐95.ResultsThe association of CD36 with caveolin‐1 is increased after stimulation with ILP. The tyrosine kinase Src kinase‐1 acts to phosphorylate dynamin‐2, a GTPase responsible for separating the vesicle from the plasma membrane. ILP stimulation induced phosphorylation of Src kinase 1 and increase in expression of dynamin2. ILP induced increased phosphorylation and Hsp90 association of eNOS, leading to increased synthesis of nitric oxide. Furthermore, the association and expression of lysosomal markers CD63 and FM5‐95 are increased after ILP treatment compared to no treatment.Summary and ConclusionOur data suggest that administration of ILP to endothelial cells induces an endosomal pathway by formation of CD36/caveolin association which leads to enhanced endosome formation seen by increased lysosomal markers. Furthermore, we can report that stimulation of endothelial cells with ILP results in an increase of eNOS activity and increased NO synthesis which might be important for the cardioprotective abilities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P.262 - Antisense oligonucleotide-mediated Dnm2 knockdown delays myotubular myopathy in mice after a single injection

Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Most recently we have shown that weekly systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. In addition, systemic weekly ASO injection into severely affected mice leads to reversal of muscle pathology within two weeks. Here we perform a single injection of ASO into 3-week-old Mtm1KO mice, and complete clinical, histological and molecular analysis of the duration of the effects after a single injection. A single injection of 25 mg/kg of ASO targeting DNM2 increased the lifespan, whole body strength, and reduced disease severity in Mtm1KO mice compared to untreated controls. Despite these results, a single injection alone was not sufficient to rescue the disease for the lifespan of the mice, suggesting repeated treatments will be required. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease.

Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice

Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Here we show that systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. Moreover, systemic ASO injection into severely affected mice leads to reversal of muscle pathology within 2 weeks. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease.

A novel AHI-1\u2013BCR-ABL\u2013DNM2 complex regulates leukemic properties of primitive CML cells through enhanced cellular endocytosis and ROS-mediated autophagy

Tyrosine kinase inhibitor (TKI) therapies induce clinical remission with remarkable effects on chronic myeloid leukemia (CML). However, very few TKIs completely eradicate the leukemic clone and persistence of leukemic stem cells (LSCs) remains challenging, warranting new, distinct targets for improved treatments. We demonstrated that the scaffold protein AHI-1 is highly deregulated in LSCs and interacts with multiple proteins, including Dynamin-2 (DNM2), to mediate TKI-resistance of LSCs. We have now demonstrated that the SH3 domain of AHI-1 and the proline rich domain of DNM2 are mainly responsible for this interaction. DNM2 expression was significantly increased in CML stem/progenitor cells; knockdown of DNM2 greatly impaired their survival and sensitized them to TKI treatments. Importantly, a new AHI-1–BCR-ABL–DNM2 protein complex was uncovered, which regulates leukemic properties of these cells through a unique mechanism of cellular endocytosis and ROS-mediated autophagy. Thus, targeting this complex may facilitate eradication of LSCs for curative therapies.

Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion.

Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway.

Targeting High Dynamin2 Expression By Restoring Ikaros Function in Acute Lymphoblastic Leukemia

Background: Dynamin-2 (DNM2) is a GTPase essential for intracellular vesicle formation and trafficking, cytokinesis and receptor endocytosis. Mutations in DNM2 are common in early T-cell precursor acute lymphoblastic leukemia (ALL). However, DNM2 expression in other types of ALL is not reported. Ikaros, encoded by IKZF1, is a transcriptor factor functioned as a tumor suppress gene, and its dysfunction is associated with poor survival and high relapse rate in ALL. Casein Kinase II (CK2) inhibition could restore Ikaros function in high-risk leukemia and CK2 inhibitor-CX4945 showed the therapeutic efficacy on high-risk leukemia with human-derived xenograft mouse model. It is still undetermined if Ikaros regulates DNM2 expression in the leukemic cells.Methods: The 151 patients’ and 30 volunteers’ BM samples were collected between June 2008 and June 2014 at the First Affiliated Hospital of Nanjing Medical University. The ALL diagnosis was made according to the morphologic, Immunophenotypic, cytogenetic, and molecular criteria of WHO Diagnosis and Classification of ALL (2008).Cytogenetic and molecular analyses as previously reported. The DNM2 expression was determined by qPCR in the patients. All the patients were divided into high or low DNM2 expression groups (Q4 vs Q1-3) and the cutoff was determined by SPSS 17.0. For quantitative parameters, overall differences between the cohorts were evaluated using a Mann - Whitney U -test. For qualitative parameters, overall group differences were analyzed using a χ2 test. All statistical analyses were performed using the SPSS 17.0 and P<0.05 was considered statistically significant. The effect of Ikaros on DNM2 gene expression was observed by qPCR in the leukemic cells expressed Ikaros or Ikaros ShRNA. Ikaros binding with promoter of DNM2 was evaluated by chromatin immunoprecipitation assay following quantitative real-time PCR in leukemic cells. The effect of DNM2 inhibitor on cell proliferation was performed by WST-1 cell proliferation assay, and the synergy of Casein Kinase inhibitor which restores Ikaros function with DNM2 inhibitor on cell proliferation of leukemic cells was analyzed by CalcuSyn.Results: We studied DNM2 mRNA level in adults with B- and T-cell ALL, and found DNM2 is more highly expressed compared with normals in both forms of ALL. High DNM2 expression is significantly associated with poor overall survival (OS), high relapse rate, and leukaemia cell proliferation markers particularly in B-ALL. DNM2 expression is significantly higher in the patients with IKZF1 deletion compared to that of without deletion. Ikaros directly binds the DNM2 promoter in Nalm6 (B-ALL) and CEM (T-ALL) leukemic cells. Ikaros suppresses the transcription of DNM2 with luciferase reporter assay. Retroviral transduction of Ikaros results in the down-regulation of DNM2 in the leukemic cells. CK2 inhibitor, TBB increases Ikaros binding to promoter of DNM2 and suppresses DNM2 expression in an Ikaros-dependent manner in both leukemic cell lines and primary cells. TBB induced-increase of H3K9me3 binding on the promoter of DNM2 was also observed in leukemic cell lines and primary cells. Finally, DNM2 inhibitor-MiTMAB significantly suppresses the cell proliferation of Nalm6 and CEM cells with the WST-1 cell proliferation assay and has significantly synergistic effect with Ck2 inhibitor, CX-4945 in the cells.Conclusion: High DNM2 expression is associated with Ikarosdys-regulation, revealing their potential roles on the development of ALL. DNM2 inhibitor MiTMAB inhibits cell proliferation and has synergistic effect with CK2 inhibitor CX4945 in leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Targeting High Dynamin-2 (DNM2) Expression by Restoring Ikaros Function in Acute Lymphoblastic Leukemia.

Dynamin-2 (DNM2) is a GTPase essential for intracellular vesicle formation and trafficking, cytokinesis and receptor endocytosis. Mutations in DNM2 are common in early T-cell precursor acute lymphoblastic leukemia. However, DNM2 expression in other types of ALL are not reported. We studied DNM2 mRNA level in adults with B- and T-cell ALL. We found DNM2 is more highly expressed compared with normals in both forms of ALL. High DNM2 expression is associated with some clinical and laboratory features, inferior outcomes and with leukaemia cell proliferation. We also found Ikaros directly binds the DNM2 promoter and suppresses DNM2 expression. Consequently IKZF1 deletion is associated with high DNM2 expression. Conversely, casein kinase-2 (CK2)-inhibitor increases Ikaros function thereby inhibiting DNM2 expression. Inhibiting DNM2 suppresses proliferation of leukemia cells and synergizes with CK2 inhibition. Our data indicate high DNM2 expression is associated with Ikaros dysregulation and may be important in the development of B-ALL.