Proliferation Of Neuroblastoma Cell Lines Research Articles

Therapeutic benefit of the dual ALK/FAK inhibitor ESK440 in ALK-driven neuroblastoma

Neuroblastoma (NB) is a predominantly pediatric cancer with greater than 90% of cases arising in children under the age of five. More than half of patients have metastases detected at diagnosis, and high-risk disease is associated with five-year survival rates of only 50–60 %. Standard therapy involves highly toxic chemotherapy, surgery, radiation, and immunotherapy, and less toxic, more specific targeted therapies are urgently needed. Genomic studies have identified common driver aberrations in high-risk NB, such as MYCN amplification. In addition, a proportion of high-risk patients harbor amplification or activating mutations in anaplastic lymphoma kinase (ALK), and co-occurrence of ALK mutations and MYCN amplification have been associated with aggressive disease. In this study, we analyzed the efficacy of a Phase Ia-cleared, orally bioavailable dual ALK and focal adhesion kinase (FAK) inhibitor, ESK440, in multiple preclinical NB models. ESK440 potently inhibited proliferation of NB cell lines, with increased sensitivity in cell lines harboring ALK aberrations. ALK, FAK, and downstream target activation were rapidly decreased upon ESK440 treatment, and this was associated with impaired cellular migration and invasion. Importantly, ESK440 treatment also decreased MYCN levels. NB cell line and patient-derived xenograft studies showed significant reduction in tumor growth in ESK440-treated mice with no signs of toxicity. In certain NB models, ESK440 showed comparable or enhanced efficacy to lorlatinib, another clinical ALK inhibitor, and a lorlatinib-resistant cell line (COG-N-561 LR) retained sensitivity to ESK440. These preclinical results indicate that ESK440 is a promising targeted agent for ALK-driven NB and support future clinical studies to evaluate its efficacy in NB patients.

Abstract 280: Targeting mRNA pseudouridylation to suppress neuroblastoma metabolism

Abstract Pseudouridylation of mRNA is a common epigenetic modification that reduces RNA conformational flexibility and increases its structural stability and may increase its availability for translation. RNA pseudouridylation is catalyzed by the family of pseudouridine synthases, including dyskerin pseudouridine synthase 1 (DKC1). Here we report the DKC1 gene is a direct transcriptional target of MYCN, which is a major oncogene in driving the development of high-risk neuroblastoma, a childhood cancer with a poor prognosis. The transcriptional upregulation of DKC1 expression in MYCN-amplified cells is of functional significance, as DKC1 depletion by RNA interference inhibits the proliferation of neuroblastoma cell lines. Furthermore, gene expression profiling reveals that DKC1 has a key role in the regulation of amino acid biosynthesis enzymes. Genetic and pharmacological inhibition of DKC1 reduces the expression of ATF4 and its downstream target genes involved in amino acid synthesis and transport. These findings reveal a critical role of DKC1 in sustaining the proliferation of neuroblastoma cells by promoting amino acid metabolism, which could be exploited as a therapeutic strategy against high-risk neuroblastoma. Citation Format: Anamika Gupta, Mohit Bansal, Han-Fei Ding. Targeting mRNA pseudouridylation to suppress neuroblastoma metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 280.

Abstract 6242: Inhibition of BIRC5/survivin by LQZ-7I inhibits neuroblastoma growth while hemizygosity of BIRC5 does not: implications for therapy of neuroblastoma

Abstract Overexpression of survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB). Transcriptional inhibitors of survivin have been tested in clinical trials. However, results have been disappointing. We thus investigated the small molecules LQZ-7I and S12 that inhibit dimerization of survivin protein and compared their growth-inhibiting efficacy with that of markedly decreased survivin expression in a novel transgenic NB mouse. Mice hemizygous for BIRC5 were crossed with TH-MYCN mice to generate BIRC5+/−/MYCNtg/+ mice. NB incidence, growth and retransplantation capacity were assessed. In vitro, LQZ-7I and S12 were used to inhibit survivin protein interactions and the effects on proliferation and cell cycle of NB cells were investigated. BIRC5 hemizygous mice developed NB although mRNA and protein expression of survivin was markedly decreased. Surprisingly, incidence and latency of the NB that developed in the BIRC5+/−/MYCNtg/+ mice did not differ from the NB in the TH-MYCN mice, and tumor growth and survival of the mice was not affected. In stark contrast, the survivin interaction inhibitors LQZ-7I and S12 abolished anchorage-independent growth and proliferation of NB cell lines. Reduced growth of NB cells resulted from defects in mitosis induced by LQZ-7I or S12 or. LQZ-7I was the more efficient inhibitor of NB cells while S12 was not able to reduce tumor growth in a xenograft model. Taken together, these results support the notion that inhibiting survivin interaction is more efficacious in NB than inhibiting survivin transcription. Citation Format: Carmen Dorneburg, Celimene Galliger, Voker Rasche, Andre Lechel, Thomas F. Barth, Klaus-Michael Debatin, Christian Beltinger. Inhibition of BIRC5/survivin by LQZ-7I inhibits neuroblastoma growth while hemizygosity of BIRC5 does not: implications for therapy of neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6242.

YY1 transcription factor induces proliferation and aerobic glycolysis of neuroblastoma cells via LDHA regulation.

The present study investigated the effect of transcription factor yin yang 1 (YY1) on aerobic glycolysis and cell proliferation in neuroblastoma and its mechanism. Neuroblastoma cell lines were used to investigate the association between YY1 and lactate dehydrogenase A (LDHA) expression. Cell Counting Kit (CCK)-8 and clone formation experiments were used to detect the cell viability. The interaction of YY1 and LDHA was detected using chromatin immunoprecipitation assay. Glucose uptake, intra/extracellular lactate and pyruvate and LDHA expression were evaluated using standard methods. Reverse transcription quantitative PCR, western blotting and gene overexpression or silencing were undertaken to explore the biological effects and underlying mechanisms of transcriptional regulators in NB cells. The results demonstrated that YY1 was significantly upregulated in neuroblastoma cell lines. The results of aerobic glycolysis and CCK-8 indicated that YY1 significantly promoted the proliferation and aerobic glycolysis of neuroblastoma cells. In addition, chromatin immunoprecipitation-PCR results demonstrated that YY1 was directly bound to the promoter LDHA. Overexpression of LDHA could reverse the inhibitory effect of sh-YY1 on aerobic glycolysis and proliferation of neuroblastoma cells. In conclusion, YY1 could induce aerobic glycolysis and proliferation of neuroblastoma cell lines, and may directly mediate the regulation of LDHA. These findings may provide novel insight for the treatment of neuroblastoma.

Abstract 411: Risk-dependent enhancer transcription defines oncogenic gene regulatory networks for high-risk neuroblastoma

Abstract Neuroblastoma is the most frequent extracranial solid tumor in childhood. High-risk neuroblastoma (HR-NB) with less than 50% five-year event-free survival rates (EFSR) is distinct from non-high-risk neuroblastoma (NHR-NB) with above 90% EFSR. Investigating risk-dependence is therefore essential, and that of noncoding regulation is extra essential because to date only few coding mutations have been found in pediatric tumors. Among noncoding regulators, active enhancer plays the most crucial role to precisely control gene expression. However, given surrogate marks of enhancers, identifying functional enhancer is challenging. We have recently developed a noncoding transcriptome-based computational approach to define functional enhancers, given that some long noncoding RNAs (lncRNAs) transcribed from enhancers are required for enhancer activity. Here, we apply risk-dependence of lncRNA expression to identify oncogenic enhancers and enhancer-associated lncRNAs, determining an oncogenic regulatory network in HR-NB. LncRNAs are often unstably expressed thus have a higher proportion of risk-dependence than coding genes. We re-analyzed the noncoding transcriptome of sequenced primary HR-NB (n=176) versus NHR-NB (n=322) samples (tumors cell content >60%) and identified 8.3k lncRNAs (3.4k up-regulated and 4.9k down-regulated) for the high-risk patients (FC>2, FDR<0.05). Similarly, from the coding transcriptome profiling of the same patients, we identified 694 up-regulated and 925 down-regulated genes. Noncoding transcriptomic risk-group dependence is a robust metric for enhancer activity. Comparing risk-dependent distal lncRNAs with that of adjacent risk-dependent genes there exists a significant correlation in their direction and magnitude (coefficient=0.70, P<2e-16). Furthermore, risk-dependent lncRNAs reside at active enhancers specific to the neural tube (VISTA, 37 out of 257 validated enhancers, empirical P<0.005). Importantly, we observed three strong oncogenic associations for these 37 “risk-dependent and tissue-specific enhancers.” 1) 70 human transcription factors (TFs) showed significant binding potency that indicates enhancer function, exampled by the tumorigenic FOXM1. These TFs significantly over-represent known oncogenes (FET P=0.01, odds=5) and tumor suppressors (P=0.0003, odds=7.5). 2) Risk-dependent target genes that remarkably over-represent the proliferation of neuroblastoma cell lines (P=0.0001, Ingenuity Pathway Analysis) indicate a directly tumorigenic impact by these risk-dependent enhancers. 3) Risk-dependent enhancer lncRNAs reside at neuroblastoma susceptibility loci more often than by chance. In summary, risk-dependence of noncoding transcripts illuminate oncogenic enhancers with tissue-specific activity and neuroblastoma susceptibility, therefore is a general facet of oncogenic enhancer activity. Citation Format: Zhezhen Wang, Ivan P. Moskowitz, John M. Cunningham, Xinan Holly Yang. Risk-dependent enhancer transcription defines oncogenic gene regulatory networks for high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 411.

Syntheses and cytotoxicity of syringolin B-based proteasome inhibitors

The concise and modular total synthesis of the bacterial natural product and irreversible proteasome inhibitor syringolin B has been achieved. This synthesis has enabled the ready preparation of three diverse, structurally modified syringolin derivatives. The actions of these compounds in inhibiting the proliferation of neuroblastoma cell lines was evaluated, and significant enhancements in potency compared to the natural product were realized.

Abstract 4685: Mechanistic guidance of ALK inhibition for the treatment of neuroblastoma

Abstract Receptor tyrosine kinases (RTKs) are important therapeutic targets in a wide variety of human diseases, especially neoplastic disorders. Several drugs that target the tyrosine kinase domains of the epidermal growth factor receptor (EGFR) or other tyrosine kinases are now in clinical use, and it has become clear that their use requires guidance from the results of genetic, biochemical and structural studies of their targets. A similar situation is emerging with another RTK-the anaplastic lymphoma kinase (ALK)-in neuroblastoma (NB), where activating mutations are therapeutic targets. In our study, inhibition of in vitro proliferation of NB cell lines was associated with abrogation of phosphorylation of ALK by PF-02341066, an orally bioavailable, ATP-competitive inhibitor of ALK and c-Met. Cell lines expressing F1174L were observed to be resistant to PF-02341066 compared to cells expressing R1275Q in vitro. Additionally, the inhibitor caused complete and sustained regression of xenografts harboring the R1275Q mutation which accounts for ∼50% of all mutations in human NB. However, the compound displayed modest tumor growth delay against xenografts harboring F1174L mutations. Little is known about the normal mechanism of activation of this kinase, and our data indicate that, under the proper conditions, classical trans-autophosphorylation of the activation loop in the recombinantly expressed tyrosine kinase domain (TKD) results in a marked (∼80 fold) increase in catalytic efficiency. Recent crystal structures of ALK-TKD have revealed, surprisingly, that autoinhibition is achieved by a mechanism similar to that of EGFR rather than other insulin receptor family members. However, ALK relieves this autoinhibition solely by autophosphorylation whereas EGFR depends on allosteric interactions between dimerized domains. Our kinetic data indicate that the R1275Q and F1174L mutations perturb autoinhibitory features of this kinase and activate the TKD to varying degrees, with the F1174L mutant displaying more dramatic dysregulation. Furthermore, F1174L displayed a lower Km, ATP value compared to R1275Q, directly analogous to the EGFR L858R/T790M resistant double mutant compared to EGFR harboring the L858R mutation alone. Additionally, F1174L was nearly identical to wild-type and R1275Q ALK in screens using a panel of commercially available inhibitors. This suggests that the reason for the relative resistance of F1174L-expressing cell lines to PF-02341066 is due primarily to stronger ATP affinity. This study provides preclinical rationale for the use of PF-02341066 in NB as well as important insight into how ALK is regulated in normal and disease states. To effectively target F1174L, an innate resistance mechanism for NB cells expressing it, as well as an acquired resistance mechanism for ALK-translocated tumors, we propose that next-generation ALK inhibitors should bind the ATP-pocket with higher affinity or irreversibly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4685. doi:10.1158/1538-7445.AM2011-4685

Clinical outcome in children with recurrent neuroblastoma treated with ABT‐751 and effect of ABT‐751 on proliferation of neuroblastoma cell lines and on tubulin polymerization in vitro

ABT-751, an orally bioavailable sulfonamide, binds beta-tubulin to inhibit microtubule polymerization. We described response and event-free survival (EFS) in children with neuroblastoma and other solid tumors receiving ABT-751, assessed in vitro cytotoxicity of ABT-751 and evaluated the effect of ABT-751 on tubulin polymerization in peripheral blood mononuclear cells (PBMC) and pediatric tumor cell lines. Patients with neuroblastoma (n = 50) or other solid tumors (n = 26) enrolled on the ABT-751 pediatric phase I and pilot trials were reviewed. The sulforhodamine B (SRB) and ACEA Real-Time Cell Electronic Sensing (RT-CES) assays were used to determine the in vitro cytotoxicity. Pharmacodynamic effects on tubulin polymerization/depolymerization were assessed by Western blot and confocal microscopy using antibodies specific for post-translational modifications of polymerized tubulin. Forty-five patients with neuroblastoma were evaluated for anti-tumor response. No complete or partial responses were documented. The median EFS was 9.3 weeks for children with neuroblastoma and 3.3 weeks for children other solid tumors (P < 0.0001). The ABT-751 IC(50) was 0.6-2.6 mcM in neuroblastoma and 0.7-4.6 mcM in other solid tumor cell lines. Following drug exposure, polymerized tubulin decreased in a concentration- and time-dependent manner in cell lines. In children treated with ABT-751, the EFS is longer in children with neuroblastoma as compared to other diagnoses. In vitro, ABT-751 was cytotoxic at concentrations tolerable in children. Effects of ABT-751 on polymerization and microtubule structure were time- and dose-dependent but not dependent on tumor type.

Protein kinase B modulates the sensitivity of human neuroblastoma cells to insulin‐like growth factor receptor inhibition

The potential of the novel insulin-like growth factor receptor (IGF-IR) inhibitor NVP-AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP-AEW541 with IC(50) values ranging from 0.15 to 5 microM. Experiments using an IGF-IR neutralizing antibody confirmed that the IGF-IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF-IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP-AEW541, while coexpression of the IGF-IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP-AEW541. Inhibition of Akt/PKB activity restored the sensitivity of neuroblastoma cells to the IGF-IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP-AEW541. IGF-I-stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP-AEW541, or by a combination of an inhibitor of phosphoinositide 3-kinase and rapamycin. In addition to its antiproliferative effects, NVP-AEW541 sensitized neuroblastoma cells to cisplatin-induced apoptosis. Together, our data demonstrate that NVP-AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation.

Proliferation of Human Neuroblastomas Mediated by the Epidermal Growth Factor Receptor

Neuroblastoma is a common solid tumor of childhood that is derived from the neural crest. Expression of epidermal growth factor (EGF) receptors (EGFRs) has been associated with enhanced cell growth and aggressive behavior in other tumors. Here, we examined the expression profile of EGFRs in neuroblastoma cell lines and primary tumors. We found that all 13 neuroblastoma cell lines examined expressed EGFR1 (HER1), most at readily detectable levels. Low levels of other human EGFR family receptors were also detected in almost all cell lines. All primary tumors examined expressed readily detectable levels of HER1 and HER3 and lower levels of HER2 and HER4. EGF had a significant effect on the proliferation of neuroblastoma cell lines in vitro. EGF treatment (100 ng/mL) of the cell lines SY5Y and NLF significantly increased cell number (P < 0.01). EGF stimulated more cells to enter S and G2-M phase, as suggested by flow cytometry, indicating that EGF increases cell number by increasing proliferation, with no appreciable change in apoptosis. EGF exposure resulted in receptor autophosphorylation and activation of both the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Exposure to 0.5 micromol/L ZD1839, a HER1-specific inhibitor, caused a 40% to 50% reduction in the number of SY5Y and NLF cells grown in medium containing 10% fetal bovine serum (P < 0.01). Even at 0.01 micromol/L, ZD1839 inhibited autophosphorylation of HER1 by EGF. At 0.1 micromol/L, it also blocked phosphorylation of AKT, but not MAPK, in NLF cells. Additional studies showed that the PI3K/AKT-specific inhibitor LY294002 had a more profound effect than the MAPK-specific inhibitor U0126 in blocking EGF-induced cell proliferation. This suggests that the PI3K/AKT pathway is the main signaling pathway responsible for the proliferation effects of EGF in neuroblastomas. Our results also indicate that ZD1839 is a potent inhibitor of neuroblastoma cell proliferation; therefore, it may be a useful, biologically based therapeutic agent for these tumors.