ALT neuroblastoma chemoresistance due to telomere dysfunction–induced ATM activation is reversible with ATM inhibitor AZD0156

2021 ◽  
Vol 13 (607) ◽  
pp. eabd5750
Author(s):  
Balakrishna Koneru ◽  
Ahsan Farooqi ◽  
Thinh H. Nguyen ◽  
Wan Hsi Chen ◽  
Ashly Hindle ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ataxia Telangiectasia ◽  
De Novo ◽  
Clinical Stage ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Telomere Dysfunction ◽  
Neuroblastoma Cell Lines

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

scholarly journals ALT Neuroblastoma Chemoresistance due to ATM Activation by Telomere Dysfunction is Reversible with the ATM Inhibitor AZD0156

2021 ◽  
Author(s):  
Balakrishna Koneru ◽  
Ahsan Farooqi ◽  
Thinhh H. Nguyen ◽  
Wan Hsi Chen ◽  
Ashly Hindle ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Clinical Stage ◽  
Neuroblastoma Cell ◽  
Telomere Dysfunction ◽  
Atm Kinase ◽  
Kinase Activation ◽  
Neuroblastoma Cell Lines

AbstractCancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ∼ 25% of high-risk neuroblastomas and relapse or progression in ALT neuroblastoma patients during or after front-line therapy is frequent and almost uniformly fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell-lines and xenografts established from relapsed ALT neuroblastoma patients demonstrated de novo resistance to temozolomide + irinotecan (as SN-38 in vitro, P<0.05) and in vivo (mouse event-free survival (EFS) P<0.0001) relative to telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifest constitutive ATM kinase activation due to spontaneous telomere dysfunction while telomerase- positive tumors lacked constitutive ATM activation or spontaneous telomere DNA damage. We demonstrated that induction of telomere dysfunction resulted in ATM activation that in turn conferred resistance to temozolomide + SN-38 (4.2 fold-change in IC50, P<0.001). ATM kinase shRNA knock-down or inhibition using a clinical-stage small molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell-lines in vitro (P<0.001) and in 4 ALT xenografts in vivo (EFS P<0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. ATR inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cell lines. Thus, resistance to chemotherapy in ALT neuroblastoma occurs via ATM kinase activation and was reversed with the ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing in neuroblastoma.One Statement SummaryATM activation at telomeres confers resistance to DNA damaging chemotherapy in ALT neuroblastoma that was reversed with ATM knockdown or inhibition.

scholarly journals Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

2021 ◽  
Vol 3 (4) ◽  
pp. 12-24
Author(s):  
Mabao YUAN ◽  
Hanjiao HANG ◽  
Lubin YAN ◽  
Xuanjie HUANG ◽  
Ziyang SANG ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Luciferase Reporter ◽  
Ubiquitin Protein Ligase ◽  
Neuroblastoma Cell Lines ◽  
Dual Luciferase Reporter Assay

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.

scholarly journals GX15–070 (Obatoclax), a Bcl-2 family proteins inhibitor engenders apoptosis and pro-survival autophagy and increases Chemosensitivity in neuroblastoma

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Sonia Cournoyer ◽  
Anissa Addioui ◽  
Assila Belounis ◽  
Mona Beaunoyer ◽  
Carine Nyalendo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Distant Metastases ◽  
Caspase Inhibitor ◽  
Pediatric Tumor ◽  
Apoptotic Protein ◽  
Synergistic Cytotoxicity ◽  
Neuroblastoma Cell Lines

Abstract Background Neuroblastoma (NB) is a frequent pediatric tumor associated with poor prognosis. The disregulation of Bcl-2, an anti-apoptotic protein, is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate therapeutic efficiency of GX 15–070, a pan-Bcl-2 family inhibitor, used alone and in combination with conventional drugs or with hydroxychloroquine (HCQ), an autophagy inhibitor. Methods Five neuroblastoma cell lines were tested for the cytotoxic activity of GX 15–070 alone or in combination with cisplatin, doxorubicin, HCQ or Z-VAD-FMK a broad-spectrum caspase inhibitor. Apoptosis and autophagy levels were studied by western-blot and FACS. Orthotopic injections were performed on NOD/LtSz-scid/IL-2Rgamma null mice that were treated with either GX 15–070 alone or in combination with HCQ. Results Synergistic cytotoxicity was observed for the drug combination in all of the 5 neuroblastoma cell lines tested, including MYCN amplified lines and in cancer stem cells. GX 15–070 significantly increased apoptosis and autophagy in neuroblastoma cells as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by HCQ, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agent plays a cytoprotective role. In vivo, GX 15–070 combined with HCQ significantly decreased the growth of the tumor and the number of distant metastases. Conclusions Based on the synergistic effect of HCQ and GX 15–070 observed in this study, the combination of these two drugs may be utilized as a new therapeutic approach for neuroblastoma.

scholarly journals Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

2010 ◽  
Vol 32 (1-2) ◽  
pp. 77-86
Author(s):  
Maria Valeria Corrias ◽  
Claudio Gambini ◽  
Andrea Gregorio ◽  
Michela Croce ◽  
Gaia Barisione ◽  
...  
Keyword(s):  
Nervous System ◽  
Subcellular Localization ◽  
Cell Lines ◽  
System Development ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Nervous System Development ◽  
Primary Tumors ◽  
Neuroblastoma Cell Lines

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system.Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively.Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype.Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up.

scholarly journals SYK Inhibition Potentiates the Effect of Chemotherapeutic Drugs on Neuroblastoma Cells in Vitro

Cancers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 202 ◽  
Author(s):  
Conny Tümmler ◽  
Gianina Dumitriu ◽  
Malin Wickström ◽  
Peter Coopman ◽  
Andrey Valkov ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inflammatory Diseases ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Chemotherapeutic Drugs ◽  
Akt Phosphorylation ◽  
Risk Patients ◽  
Syk Inhibitor ◽  
Neuroblastoma Cell Lines

Neuroblastoma is a malignancy arising from the developing sympathetic nervous system and the most common and deadly cancer of infancy. New therapies are needed to improve the prognosis for high-risk patients and to reduce toxicity and late effects. Spleen tyrosine kinase (SYK) has previously been identified as a promising drug target in various inflammatory diseases and cancers but has so far not been extensively studied as a potential therapeutic target in neuroblastoma. In this study, we observed elevated SYK gene expression in neuroblastoma compared to neural crest and benign neurofibroma. While SYK protein was detected in the majority of examined neuroblastoma tissues it was less frequently observed in neuroblastoma cell lines. Depletion of SYK by siRNA and the use of small molecule SYK inhibitors significantly reduced the cell viability of neuroblastoma cell lines expressing SYK protein. Moreover, SYK inhibition decreased ERK1/2 and Akt phosphorylation. The SYK inhibitor BAY 613606 enhanced the effect of different chemotherapeutic drugs. Transient expression of a constitutive active SYK variant increased the viability of neuroblastoma cells independent of endogenous SYK levels. Collectively, our findings suggest that targeting SYK in combination with conventional chemotherapy should be further evaluated as a treatment option in neuroblastoma.

scholarly journals Anti-cancer effects of bortezomib against chemoresistant neuroblastoma cell lines in vitro and in vivo

2006 ◽  
Author(s):  
Martin Michaelis ◽  
Iduna Fichtner ◽  
Diana Behrens ◽  
Wolfram Haider ◽  
Florian Rothweiler ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Anti Cancer ◽  
Neuroblastoma Cell Lines

scholarly journals Down-Regulation of Phosphoribosyl Pyrophosphate Synthetase 1 Inhibits Neuroblastoma Cell Proliferation

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 955
Author(s):  
Jifu Li ◽  
Junhong Ye ◽  
Shunqin Zhu ◽  
Hongjuan Cui
Keyword(s):  
Cell Proliferation ◽  
De Novo ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Phosphoribosyl Pyrophosphate ◽  
Down Regulation ◽  
Nucleotide Synthesis ◽  
Phosphoribosyl Pyrophosphate Synthetase

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.

scholarly journals Specific Inhibition of GSK-3β by Tideglusib: Potential Therapeutic Target for Neuroblastoma Cancer Stem Cells

2020 ◽  
Author(s):  
Hisham F. Bahmad ◽  
Reda M. Chalhoub ◽  
Hayat Harati ◽  
Jolie Bou-Gharios ◽  
Farah Ballout ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
And Migration ◽  
Gsk 3Β ◽  
Neuroblastoma Cell Lines

AbstractNeuroblastoma is an embryonic tumor that represents the most common extracranial solid tumor in children. Resistance to therapy is attributed, in part, to the persistence of a subpopulation of slowly dividing cancer stem cells (CSCs) within those tumors. Glycogen synthase kinase (GSK)-3β is an active proline-directed serine/threonine kinase, well-known to be involved in different signaling pathways entangled in the pathophysiology of neuroblastoma. This study aims to assess the potency of an irreversible GSK-3β inhibitor drug, Tideglusib (TDG), in suppressing proliferation, viability, and migration of human neuroblastoma cell lines, as well as its effects on their CSCs subpopulation in vitro and in vivo. Our results showed that treatment with TDG significantly reduced cell proliferation, viability, and migration of SK-N-SH and SH-SY5Y cells. TDG also significantly inhibited neurospheres formation capability in both cell lines, eradicating the self-renewal ability of highly resistant CSCs. Importantly, TDG potently inhibited neuroblastoma tumor growth and progression in vivo. In conclusion, TDG proved to be an effective in vitro and in vivo treatment for neuroblastoma cell lines and may hence serve as a potential adjuvant therapeutic agent for this aggressive nervous system tumor.

scholarly journals EZH2 inhibition decreases neuroblastoma proliferation and in vivo tumor growth

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0246244
Author(s):  
Laura V. Bownes ◽  
Adele P. Williams ◽  
Raoud Marayati ◽  
Laura L. Stafman ◽  
Hooper Markert ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Migration And Invasion ◽  
Human Neuroblastoma ◽  
Cancer Types ◽  
Neuroblastoma Cell Lines

Investigation of the mechanisms responsible for aggressive neuroblastoma and its poor prognosis is critical to identify novel therapeutic targets and improve survival. Enhancer of Zeste Homolog 2 (EZH2) is known to play a key role in supporting the malignant phenotype in several cancer types and knockdown of EZH2 has been shown to decrease tumorigenesis in neuroblastoma cells. We hypothesized that the EZH2 inhibitor, GSK343, would affect cell proliferation and viability in human neuroblastoma. We utilized four long-term passage neuroblastoma cell lines and two patient-derived xenolines (PDX) to investigate the effects of the EZH2 inhibitor, GSK343, on viability, motility, stemness and in vivo tumor growth. Immunoblotting confirmed target knockdown. Treatment with GSK343 led to significantly decreased neuroblastoma cell viability, migration and invasion, and stemness. GSK343 treatment of mice bearing SK-N-BE(2) neuroblastoma tumors resulted in a significant decrease in tumor growth compared to vehicle-treated animals. GSK343 decreased viability, and motility in long-term passage neuroblastoma cell lines and decreased stemness in neuroblastoma PDX cells. These data demonstrate that further investigation into the mechanisms responsible for the anti-tumor effects seen with EZH2 inhibitors in neuroblastoma cells is warranted.

scholarly journals Cell Line-Dependent Variability of Coordinate Expression of p75NTR and CRABP1 and Modulation of Effects of Fenretinide on Neuroblastoma Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yaoli Pu Yang ◽  
Simeng Wang ◽  
Xingguo Li ◽  
Nina F. Schor
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neurotrophin Receptor ◽  
Human Neuroblastoma Cell ◽  
P75 Neurotrophin Receptor ◽  
Human Neuroblastoma ◽  
Neuroblastoma Cell Lines

Neuroblastoma is a childhood neural crest tumor. Fenretinide, a retinoic acid analogue, induces accumulation of mitochondrial reactive oxygen species and consequent apoptosis in neuroblastoma cells. The p75 neurotrophin receptor (p75NTR) enhances the antineuroblastoma cell efficacy of fenretinidein vitro. We examined the role of the retinoid binding protein, CRABP1, in p75NTR-mediated potentiation of the efficacy of fenretinide. Knockdown and overexpression, respectively, of either p75NTR or CRABP1 were effected in neuroblastoma cell lines using standard techniques. Expression was determined by qRT-PCR and confirmed at the protein level by Western blot. Metabolic viability was determined by Alamar blue assay. While protein content of CRABP1 correlated roughly with that of p75NTR in the three neuroblastoid or epithelioid human neuroblastoma cell lines studied, manipulation of p75NTR expression resulted in cell line-dependent, variable change in CRABP1 expression. Furthermore, in some cell lines, induced expression of CRABP1 in the absence of p75NTR did not alter cell sensitivity to fenretinide treatment. The effects of manipulation of p75NTR expression on CRABP1 expression and the effects of CRABP1 expression on fenretinide efficacy are therefore neuroblastoma cell line-dependent. Potentiation of the antineuroblastoma cell effects of fenretinide by p75NTR is not mediated solely through CRABP1.

Sign in / Sign up

Export Citation Format

Share Document