Abstract
G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and ...cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound.
Lysosomes play a key role in regulating cell death in response to cancer therapies, yet little is known on the possible role of lysosomes in the therapeutic efficacy of G-quadruplex DNA ligands (G4L) ...in cancer cells. Here, we investigate the relationship between the modulation of lysosomal membrane damage and the degree to which cancer cells respond to the cytotoxic effects of G-quadruplex ligands belonging to the triarylpyridine family. Our results reveal that the lead compound of this family, 20A promotes the enlargement of the lysosome compartment as well as the induction of lysosome-relevant mRNAs. Interestingly, the combination of 20A and chloroquine (an inhibitor of lysosomal functions) led to a significant induction of lysosomal membrane permeabilization coupled to massive cell death. Similar effects were observed when chloroquine was added to three new triarylpyridine derivatives. Our findings thus uncover the lysosomal effects of triarylpyridines compounds and delineate a rationale for combining these compounds with chloroquine to increase their anticancer effects.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
G-quadruplex (G4) ligands have shown anti-tumor activity by stabilizing G4 structures. We recently reported that the G4 ligand 20A promotes senescence in cancer cells through Ataxia Telangiectasia ...Mutated (ATM) and autophagy activation. Inhibition of both pathways directs cells to apoptosis, positioning ATM/autophagy axis as a linchpin between senescence and apoptosis.
Background: Myelodysplastic syndromes (MDS) are a heterogenous group of stem cell driven disorders primarily affecting the elderly and characterized by inefficient production of mature blood cells ...and a high risk (30%) of evolution to secondary acute myeloid leukemia. Despite tremendous progress in the past decade, treatment options for MDS patients remain limited, and primarily address disease symptoms, rather than altering disease course. This points to the urgent need to better understand the pathogenesis of this heterogenous group of syndromes to develop new therapies that address disease vulnerabilities. However, this effort has been largely hampered by the limited availability of model systems that allow the exploration of MDS biology in a fully humanized setting. In recent years, studies from our lab and others, have highlighted the crucial role niche cells play in human MDS, hence reinforcing the notion that MDS is a disease of a tissue rather than hematopoietic cells alone. Therefore, exploration of MDS biology requires the further development of fully human MDS models in which both constituents of the disease, namely hematopoietic and niche cells, are present.
Methods: To address this issue we successfully isolated endothelial cells (ECs) and mesenchymal stromal cells (MSC) from bone marrow biopsies obtained from MDS patients or healthy age matched controls, and subsequently utilized them to develop fully human 2D and 3D organotypic niche models, which were successfully used to support normal and MDS HSPCs expansion ex-vivo. The 3D system makes use of a collagen scaffold, as this protein makes up for 90% of the matrix proteins in the bone. Importantly, MSC and EC cultures could be successfully established from several independent donors and immortalized to generate primary cell lines that can be used to reproducibly establish these ex-vivo systems in a robust manner. Moreover, we could show that these niche cells were easily amenable to genetic editing using CRISPR-Cas9 technology as well as modified to carry fluorescent reporter proteins for tracking cellular interactions using live cell imaging and confocal microscopy.
Results: In this work, we successfully isolated human mesenchymal and endothelial cells, from primary bone marrow biopsies (MDS and healthy) and established fully human 2D and 3D organotypic co-cultures ex-vivo. Of note, although bone marrow ECs represent an essential component of the hematopoietic niche, they have so far been omitted in previously described human bone marrow niche models, owing to the notorious difficulties in isolating and expanding this cell type from primary bone marrow biopsies. Therefore, we established immortalized EC lines (iECs) that faithfully recapitulate the morphological, phenotypic and functional features of primary bone marrow ECs. When cultured at defined ratios and under defined conditions, MSCs instructed ECs and iECs to form of vessel-like structures that mimic the meshwork observed in vivo and are typically escheated by aSMA positive cells that stabilize the structures. Genetic manipulation of the cellular components of the niche also allowed to explore the functional relevance of a specific ECM protein, which we previously identified to be significantly upregulated in MSCs isolated from MDS patients, namely the Secreted Protein Acidic and Rich in Cysteine (SPARC). SPARC ablation triggered enhanced proliferation of MDS derived HSPCs and sensitized them treatment with 5-Azacytidine, a standard of care hypomethylating agent used for the treatment of MDS patients. Additional studies are underway to further understand the underlying molecular mechanisms and define a potential druggable target that could sensitize MDS cells to standard of care treatment. Besides gene targeting studies, these organotypic models are also being used to evaluate the relative fitness of MDS and healthy stem/progenitor cells in healthy versus patient derived niches, to explore the contribution of niche components to the establishment of the progressive clonal dominance observed in MDS.
Bönig:Terumo BCT: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Kiadis: Honoraria; Bayer: Research Funding; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Fresenius: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Uniqure: Research Funding; medac: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Genzyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Healthineers: Current equity holder in publicly-traded company; Chugai: Honoraria, Research Funding; Erydel: Research Funding; Miltenyi: Honoraria, Research Funding; Polyphor: Research Funding; Sandor-Hexal: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Stage: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Platzbecker:Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Götze:Celgene: Research Funding. Medyouf:Bergenbio: Consultancy, Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The support of hematopoiesis declines with age and it was previously shown that aging hematopoietic cells show cytoskeletal deregulation. Also, efficient autophagy is required to maintain cellular ...functions with age. Here, we describe a new mouse model with reduced support of hematopoiesis and age-related cytoskeletal changes in which the Wnt5a gene is deleted in Osterix (SP7)+ niche cells (O5AD/D mice). Considering that in these mice Wnt5a is deleted in niche cells, we here investigate niche cells from O5AD/D and control mice treated with 5-fluorouracil (5FU).
We found that multipotent stromal cells (MSC) from the bone marrow of 5FU-treated O5AD/D mice show reduced CFU-F frequencies with preferential adipogenic differentiation. In these cells, the F-actin network is disorganized and associated with an elevated activity of the actin assembly-stimulating CDC42. Moreover, we found more and perinuclear accumulated ATG7+ and LC3+ autophagosomes in O5AD/D MSC. Also, LAMP1 expression was elevated and lysosomal diameters increased. But, we found no colocalization of LC3 and LAMP1 in O5AD/D MSC and concomitant autophagic flux, suggesting a defect in autophagy.
Since in deregulated MSC from 5FU-treated O5AD/D mice CDC42 activity was increased and protein location was different, we additionally treated these mice with the CDC42 inhibitor CASIN. This treatment restored the CDC42 localization in O5AD/D MSCs and their F-actin stress fibers. Furthermore, LC3 and LAMP1 colocalization, mitochondrial and lysosomal diameters, and autophagic flux were restored. Most importantly, CASIN treatment restored the presence of the hematopoietic system and rescued the ability of O5AD/D mice to survive serial 5FU treatments.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Les G-quadruplexes (G4) sont des structures non canoniques des acides nucléiques qui peuvent être formés dans des régions d’ADN ou d’ARN riches en guanines. Les ligands G4 (LG4), sont des molécules ...capables d’interagir et de stabiliser les structures G4, qui présentent de nombreuses propriétés anti-cancéreuses. Nous avons travaillé avec le LG4 20A, appartenant à la famille des triarylpyridines, qui stabilise efficacement les structures G4 in vitro. Les objectifs de ce travail ont été de déterminer les mécanismes moléculaires et cellulaires responsables des effets anti-prolifératifs du 20A dans des cellules cancéreuses. Dans cette étude, nous avons montré que le 20A induit un arrêt de la croissance cellulaire de cellules en culture et dans un modèle de xénogreffe tumorale, grâce à l’induction de la sénescence et de la mort cellulaire par apoptose. Ces réponses sont associées à l’activation de la voie des réponses aux dommages à l’ADN (DDR) via la kinase ATM, qui favorise l’autophagie (un processus catabolique) et la sénescence, tout en protégeant les cellules de l’apoptose. De plus, nous avons observé que le 20A induit un échec de la cytokinèse, conduisant à l’accumulation de cellules binucléées qui présentent une résistance à la mort cellulaire. De façon inattendue, nous avons trouvé que le 20A s’accumule dans les lysosomes, induisant une augmentation de la taille de ces derniers. La combinaison du 20A et de l’agent lysomotropique chloroquine, potentialise de façon importante la perméabilisation de la membrane lysosomale (LMP) et la mort cellulaire. En particulier, cette combinaison sensibilise de façon notable ces cellules binucléées à la mort cellulaire. L’ensemble de ces résultats révèle une relation entre les processus de mort cellulaire et de sénescence induits par le LG4 20A, et les voies de DDR et lysosomales. Ces régulations devraient être prises en considération lors de l’utilisation d’agents antiprolifératifs susceptibles d’interférer avec les fonctions lysosomales.
G-quadruplexes (G4) are unusual nucleic acid structures that can be formed by guanine-rich DNA and RNA. Through their ability to stabilize G4 structures, G4 ligands (G4L) have been described to display potent anticancer properties. Here, we studied the G4L 20A belonging to the triarylpyridine family of compounds that have the ability to efficiently bind to and stabilize G4 structures in vitro. The objectives of this work were to determine the molecular and cellular mechanisms responsible for the anti-proliferative effects of 20A in cancer cells. In this study, we showed that 20A causes cancer cell growth arrest in cell culture and a mice tumour xenograft model, through induction of senescence and apoptotic cell death. These cellular responses are associated with the induction of the DNA damage response pathway (DDR), in particular ATM activation, which promotes the induction of both autophagy (a lysosomal catabolic pathway) and senescence, while protecting cells against apoptosis. Furthermore, we found that 20A induces failure of cytokinesis which results in the accumulation of binucleated cells that display marked resistance to 20A-induced cell death. Unexpectedly, we found that 20A accumulates in the lysosomal compartment and causes lysosome enlargement. The combination of a lysosomotropic agent, chloroquine, and 20A promotes a significant induction of lysosomal membrane permeabilization (LMP) and a robust cell death. In particular, this combination significantly sensitizes binucleated cells to cell death. Altogether, our results uncover the relationship of the DDR and lysosomal pathways to cell death and senescence induced by the G4L 20A. Such regulation should also be taken into account when using antiproliferative drugs susceptible to interfere with the lysosomal functions.
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