Dystonin loss-of-function leads to impaired autophagy-endolysosomal pathway dynamics

The neuronal dystonin protein (DST-a) is a large cytoskeletal linker important for integrating the various components of the cytoskeleton. Recessive Dst mutations lead to a sensory neuropathy in mice known as dystonia musculorum (Dstdt). The disease is characterized by ataxia, autonomic disturbances, and ultimately death, which are associated with massive dorsal root ganglion (DRG) sensory neuron degeneration. Recent investigation of Dstdt sensory neurons revealed an accumulation in autophagosomes and a disruption in autophagic flux, which was believed to be due to insufficient motor protein availability. Motor protein levels and the endolysosomal pathway were assessed in pre-symptomatic (postnatal day 5; P5) and symptomatic (P15) stage wild type and Dstdt DRGs. Levels of mRNA encoding molecular motors are reduced, although no significant reduction protein level is detected. An increase in lysosomal marker LAMP1 in medium-large size Dstdt-27J sensory neurons is observed, along with an accumulation of electron-light single-membraned vesicles in Dstdt-27J DRG tissue at late stages of disease. These vesicles are likely to be autolysosomes, and their presence in only late stage Dstdt-27J sensory neurons is suggestive of a pathological defect in autophagy. Further investigation is necessary to confirm vesicle identity, and to determine the role of Dst-a in normal autophagic flux.

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p62 acts as an oncogene and is targeted by miR-124-3p in glioma

Cancer Cell International ◽

10.1186/s12935-019-1004-x ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Danni Deng ◽

Kaiming Luo ◽

Hongmei Liu ◽

Xichen Nie ◽

Lian Xue ◽

...

Keyword(s):

Glioma Cells ◽

Nuclear Factor Κb ◽

Autophagic Flux ◽

Nuclear Factor ◽

Oxygen Species ◽

Human Glioma ◽

Protein Levels ◽

Novel Therapeutic

Abstract Background Glioma is the most common central nervous system (CNS) tumour. p62, an important autophagy adaptor, plays a crucial role in cancer. However, the role of p62 in the progression of glioma is poorly characterized. Methods We examined the expression of p62 in glioma tissues and cell lines. Then we investigated the function of p62 in vitro, and clarified the mechanism underlying the regulation of p62 expression. Results We revealed that p62 was upregulated at both the mRNA and protein levels in human glioma tissues irrelevant to isocitrate dehydrogenase (IDH) status. Then, we found that overexpression of p62 promoted glioma progression by promoting proliferation, migration, glycolysis, temozolomide (TMZ) resistance and nuclear factor κB (NF-κB) signalling pathway, and repressing autophagic flux and reactive oxygen species (ROS) in vitro. In accordance with p62 overexpression, knockdown of p62 exerted anti-tumour effects in glioma cells. Subsequently, we demonstrated that miR-124-3p directly targeted the 3′-UTR of p62 mRNA, leading to the downregulation of p62. Finally, we found that p62 function could be partially reversed by miR-124-3p overexpression. Conclusions Our results demonstrate that p62 can be targeted by miR-124-3p and acts as an oncogene in glioma, suggesting the potential value of p62 as a novel therapeutic target for glioma.

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Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽

10.1182/blood.v114.22.3470.3470 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3470-3470

Author(s):

Daniela Cilloni ◽

Monica Pradotto ◽

Francesca Messa ◽

Francesca Arruga ◽

Enrico Bracco ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Western Blot ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Small Gtpases ◽

Loss Of Function ◽

Imatinib Resistance ◽

Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

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Delineating CDK9 Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Blood ◽

10.1182/blood-2021-149254 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1598-1598

Author(s):

Osman Aksoy ◽

Judith Lind ◽

Vincent Sunder-Plassmann ◽

Martin Percherstorfer ◽

Sonia Vallet ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Microenvironment ◽

Tumor Cell ◽

Treatment Strategies ◽

Loss Of Function ◽

Rna Pol Ii ◽

Pol Ii ◽

Protein Levels ◽

Molecular Events

Abstract Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, but not other genes (e.g. Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 induced a reduction of mRNA/ protein levels of Mcl-1, cMyc, RNA Pol II, but not of other potential targets (e.g. Bcl-2) in a dose- and time-dependent manner. Moreover, Thal-sns-032 reduced Mdm2 and thereby increased p53 protein levels. Consequently, Thal-sns-032 inhibited tumor cell proliferation and survival both in tumor cell- and tumor cell:BMSC co- cultures. Rationally, derived combination strategies of Thal-sns-032 for example with venetoclax, but also other investigational and established MM therapies induced synergistic anti-MM effects within the tumor microenvironment. Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derive MM combination treatment strategies. Disclosures Vallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

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UBC9-Mediated Sumoylation Favorably Impacts Cardiac Function in Compromised Hearts

Circulation Research ◽

10.1161/circresaha.115.308268 ◽

2016 ◽

Vol 118 (12) ◽

pp. 1894-1905 ◽

Cited By ~ 29

Author(s):

Manish K. Gupta ◽

Patrick M. McLendon ◽

James Gulick ◽

Jeanne James ◽

Kamel Khalili ◽

...

Keyword(s):

Cardiac Function ◽

Protein Quality ◽

Neonatal Rat ◽

Autophagic Flux ◽

Loss Of Function ◽

Specific Expression ◽

Cardiac Stress ◽

Ventricular Cardiomyocytes

Rationale: SUMOylation plays an important role in cardiac function and can be protective against cardiac stress. Recent studies show that SUMOylation is an integral part of the ubiquitin proteasome system, and expression of the small ubiquitin–like modifier (SUMO) E2 enzyme UBC9 improves cardiac protein quality control. However, the precise role of SUMOylation on other protein degradation pathways, particularly autophagy, remains undefined in the heart. Objective: To determine whether SUMOylation affects cardiac autophagy and whether this effect is protective in a mouse model of proteotoxic cardiac stress. Methods and Results: We modulated expression of UBC9, a SUMO E2 ligase, using gain- and loss-of-function in neonatal rat ventricular cardiomyocytes. UBC9 expression seemed to directly alter autophagic flux. To confirm this effect in vivo, we generated transgenic mice overexpressing UBC9 in cardiomyocytes. These mice have an increased level of SUMOylation at baseline and, in confirmation of the data obtained from neonatal rat ventricular cardiomyocytes, demonstrated increased autophagy, suggesting that increased UBC9-mediated SUMOylation is sufficient to upregulate cardiac autophagy. Finally, we tested the protective role of SUMOylation-mediated autophagy by expressing UBC9 in a model of cardiac proteotoxicity, induced by cardiomyocyte-specific expression of a mutant α-B-crystallin, mutant CryAB (CryAB R120G ), which shows impaired autophagy. UBC9 overexpression reduced aggregate formation, decreased fibrosis, reduced hypertrophy, and improved cardiac function and survival. Conclusions: The data showed that increased UBC9-mediated SUMOylation is sufficient to induce relatively high levels of autophagy and may represent a novel strategy for increasing autophagic flux and ameliorating morbidity in proteotoxic cardiac disease.

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Twitchy, the Drosophila orthologue of the ciliary gating protein FBF1/dyf-19, is required for coordinated locomotion and male fertility

Biology Open ◽

10.1242/bio.058531 ◽

2021 ◽

Author(s):

Suzanne H Hodge ◽

Amy Watts ◽

Richard Marley ◽

Richard A Baines ◽

Ernst Hafen ◽

...

Keyword(s):

Sensory Neurons ◽

Primary Cilia ◽

Loss Of Function ◽

Transmembrane Receptors ◽

C Elegans ◽

Male Germline ◽

Sensory Cilia ◽

Import And Export ◽

Flagellar Axoneme

Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. These are required for the assembly of the cilium, its function as a sensory and signalling centre and to maintain its distinctive composition. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalized primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. Here, we describe phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. Twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalized from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells.

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Role of AMPK/mTOR-independent autophagy in clear cell renal cell carcinoma

Journal of Investigative Medicine ◽

10.1136/jim-2020-001524 ◽

2020 ◽

Vol 68 (8) ◽

pp. 1386-1393

Author(s):

Milan Radovanovic ◽

Sasenka Vidicevic ◽

Jelena Tasic ◽

Nina Tomonjic ◽

Zeljka Stanojevic ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Clear Cell ◽

Tumor Stage ◽

Autophagic Flux ◽

Mrna Levels ◽

Cell Renal Cell Carcinoma ◽

Protein Levels

We examined the status and role of autophagy, a process of lysosomal recycling of cellular material, in clear cell renal cell carcinoma (ccRCC). Paired samples of tumor and adjacent non-malignant tissue were collected from 20 patients with ccRCC after radical nephrectomy. The mRNA levels of apoptosis (BAD, BAX, BCL2, BCLXL, BIM) and autophagy (ATG4, BECN1, GABARAP, p62, UVRAG) regulators were measured by RT-qPCR. The protein levels of autophagosome-associated LC3-II, autophagy receptor p62, apoptotic marker PARP, as well as phosphorylation of autophagy initiator Unc 51-like kinase 1 (ULK1), its activator AMP-activated protein kinase (AMPK) and 4EBP1, the substrate of ULK1 inhibitor mechanistic target of rapamycin (mTOR), were analyzed by immunoblotting. The mRNA levels of pro-apoptotic BAX, anti-apoptotic BCLXL and pro-autophagic ATG4, p62 and UVRAG were higher in ccRCC tumors. Autophagy induction was confirmed by an increase in phospho-ULK1 and degradation of the autophagic target p62, while apoptotic PARP cleavage was unaltered. AMPK phosphorylation was reduced and 4EBP1 phosphorylation was increased in ccRCC tissue. The expression of apoptosis regulators did not correlate with clinicopathological features of ccRCC. Conversely, high mRNA levels of ATG4, GABARAP and p62 were associated with lower tumor stage, as well as with smaller tumor size and better disease-specific 5-year survival (ATG4 and p62). Accordingly, low p62 protein levels, corresponding to increased autophagic flux, were associated with lower tumor stage, reduced metastasis and improved 5-year survival. These data demonstrate that transcriptional induction of autophagy in ccRCC is accompanied by AMPK/mTOR-independent increase in ULK1 activation and autophagic flux, which might slow tumor progression and metastasis independently of apoptosis.

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